EGFR nucleic acids and uses thereof

ABSTRACT

Disclosed herein are molecules and pharmaceutical compositions that mediate RNA interference against EGFR. Also described herein include methods for treating a disease or disorder that comprises a molecule or a pharmaceutical composition that mediate RNA interference against EGFR.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No. 62/317,105, filed Apr. 1, 2016, which application is incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 24, 2017, is named 45532-709_201_SL.txt and is 554,437 bytes in size.

BACKGROUND OF THE DISCLOSURE

Gene suppression by RNA-induced gene silencing provides several levels of control: transcription inactivation, small interfering RNA (siRNA)-induced mRNA degradation, and siRNA-induced transcriptional attenuation. In some instances, RNA interference (RNAi) provides long lasting effect over multiple cell divisions. As such, RNAi represents a viable method useful for drug target validation, gene function analysis, pathway analysis, and disease therapeutics.

SUMMARY OF THE DISCLOSURE

Disclosed herein, in certain embodiments, are molecules and pharmaceutical compositions for modulating EGFR function and/or expression in a cell.

Disclosed herein, in certain embodiments, is a polynucleic acid molecule that mediates RNA interference against EGFR, wherein the polynucleic acid molecule comprises at least one 2′ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety.

In some embodiments, the at least one 2′ modified nucleotide comprises 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified nucleotide. In some embodiments, the at least one 2′ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA). In some embodiments, the at least one inverted basic moiety is at at least one terminus. In some embodiments, the at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage.

In some embodiments, the polynucleic acid molecule is at least from about 10 to about 30 nucleotides in length. In some embodiments, the polynucleic acid molecule is at least one of: from about 15 to about 30, from about 18 to about 25, form about 18 to about 24, from about 19 to about 23, or from about 20 to about 22 nucleotides in length. In some embodiments, the polynucleic acid molecule is at least about 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

In some embodiments, the polynucleic acid molecule comprises at least one of: from about 5% to about 100% modification, from about 10% to about 100% modification, from about 20% to about 100% modification, from about 30% to about 100% modification, from about 40% to about 100% modification, from about 50% to about 100% modification, from about 60% to about 100% modification, from about 70% to about 100% modification, from about 80% to about 100% modification, and from about 90% to about 100% modification.

In some embodiments, the polynucleic acid molecule comprises at least one of: from about 10% to about 90% modification, from about 20% to about 90% modification, from about 30% to about 90% modification, from about 40% to about 90% modification, from about 50% to about 90% modification, from about 60% to about 90% modification, from about 70% to about 90% modification, and from about 80% to about 100% modification.

In some embodiments, the polynucleic acid molecule comprises at least one of: from about 10% to about 80% modification, from about 20% to about 80% modification, from about 30% to about 80% modification, from about 40% to about 80% modification, from about 50% to about 80% modification, from about 60% to about 80% modification, and from about 70% to about 80% modification.

In some embodiments, the polynucleic acid molecule comprises at least one of: from about 10% to about 70% modification, from about 20% to about 70% modification, from about 30% to about 70% modification, from about 40% to about 70% modification, from about 50% to about 70% modification, and from about 60% to about 70% modification.

In some embodiments, the polynucleic acid molecule comprises at least one of: from about 10% to about 60% modification, from about 20% to about 60% modification, from about 30% to about 60% modification, from about 40% to about 60% modification, and from about 50% to about 60% modification.

In some embodiments, the polynucleic acid molecule comprises at least one of: from about 10% to about 50% modification, from about 20% to about 50% modification, from about 30% to about 50% modification, and from about 40% to about 50% modification.

In some embodiments, the polynucleic acid molecule comprises at least one of: from about 10% to about 40% modification, from about 20% to about 40% modification, and from about 30% to about 40% modification.

In some embodiments, the polynucleic acid molecule comprises at least one of: from about 10% to about 30% modification, and from about 20% to about 30% modification.

In some embodiments, the polynucleic acid molecule comprises from about 10% to about 20% modification.

In some embodiments, the polynucleic acid molecule comprises from about 15% to about 90%, from about 20% to about 80%, from about 30% to about 70%, or from about 40% to about 60% modifications.

In some embodiments, the polynucleic acid molecule comprises at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modification.

In some embodiments, the polynucleic acid molecule comprises at least about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modifications.

In some embodiments, the polynucleic acid molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modified nucleotides.

In some embodiments, the polynucleic acid molecule comprises a sequence that hybridizes to a target sequence selected from SEQ ID NOs: 1-376.

In some embodiments, the polynucleic acid molecule comprises a single strand.

In some embodiments, the polynucleic acid molecule comprises two or more strands.

In some embodiments, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule.

In some embodiments, the second polynucleotide comprises at least one modification.

In some embodiments, the first polynucleotide and the second polynucleotide are RNA molecules. In some embodiments, the first polynucleotide and the second polynucleotide are siRNA molecules.

In some embodiments, the first polynucleotide comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 377-1892. In some embodiments, the first polynucleotide consists of a sequence selected from SEQ ID NOs: 377-1892. In some embodiments, the second polynucleotide comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 377-1892. In some embodiments, the second polynucleotide consists of a sequence selected from SEQ ID NOs: 377-1892.

Disclosed herein, in certain embodiments, is a pharmaceutical composition comprising: a) a molecule disclosed above; and b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated as a nanoparticle formulation. In some embodiments, the pharmaceutical composition is formulated for parenteral, oral, intranasal, buccal, rectal, or transdermal administration.

Disclosed herein, in certain embodiments, is a method of treating a disease or disorder in a patient in need thereof, comprising administering to the patient a composition comprising a molecule disclosed above. In some embodiments, the disease or disorder is a cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer comprises an EGFR-associated cancer. In some embodiments, the cancer comprises bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, glioblastoma multiforme, head and neck cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, or thyroid cancer. In some embodiments, the cancer comprises acute myeloid leukemia, CLL, DLBCL, or multiple myeloma.

Disclosed herein, in certain embodiments, is a method of inhibiting the expression of a target gene in a primary cell of a patient, comprising administering a molecule disclosed above to the primary cell. In some embodiments, the method is an in vivo method. In some embodiments, the patient is a human.

Disclosed herein, in certain embodiments, is a kit comprising a molecule disclosed above.

DETAILED DESCRIPTION OF THE DISCLOSURE

Epidermal growth factor receptor (EGFR, ErbB-1, or HER1) is a transmembrane tyrosine kinase receptor and a member of the ErbB family of receptors, which also include HER2/c-neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4). In some instances, EGFR mutations drive the downstream activation of RAS/RAF/MAPK, PI3K/AKT, and/or JAK/STAT pathways, leading to mitosis, cell proliferation, and suppression of apoptosis. In addition, amplification of wild-type EGFR gene has been implicated in the development of cancers such as glioblastomas and non-small cell lung cancer (Talasila, et al., “EGFR Wild-type Amplification and Activation Promote Invasion and Development of Glioblastoma Independent of Angiogenesis,” Acta Neuropathol. 125(5): 683-698 (2013); Bell et al., “Epidermal Growth Factor Receptor Mutations and Gene Amplification in Non-Small-Cell Lung Cancer: Molecular Analysis of the IDEAL/INTACT Gefitinib Trials,” J. Clinical Oncology 23(31): 8081-8092 (2005)).

Disclosed herein, in certain embodiments, are polynucleic acid molecules and pharmaceutical compositions that modulate the expression of EGFR. In some instances, the polynucleic acid molecules and pharmaceutical compositions modulate the expression of wild type EGFR gene. In other instances, the polynucleic acid molecules and pharmaceutical compositions modulate the expression of mutant EGFR.

In some embodiments, the polynucleic acid molecules and pharmaceutical compositions are used for the treatment of a disease or disorder (e.g., cancer or an EGFR-associated disease or disorder). In additional embodiments, the polynucleic acid molecules and pharmaceutical compositions are used for inhibiting the expression of EGFR gene in a primary cell of a patient in need thereof.

In additional cases, also included herein are kits that comprise one or more of polynucleic acid molecules and pharmaceutical compositions described herein.

Polynucleic Acid Molecule

In some embodiments, a polynucleic acid molecule described herein modulates the expression of the EGFR gene (GenBank: BC094761.1). In some embodiments, EGFR DNA or RNA is wild type EGFR or EGFR comprising a mutation. In some instances, EGFR is wild type EGFR. In some instances, EGFR DNA or RNA comprises a mutation. In some instances, the polynucleic acid molecule hybridizes to a target region of wild type EGFR DNA or RNA. In some instances, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising a mutation (e.g., a substitution, a deletion, or an addition).

In some instances, EGFR DNA or RNA comprises one or more mutations. In some embodiments, EGFR DNA or RNA comprises one or more mutations within one or more exons. In some instances, the one or more exons comprise exon 18, exon 19, exon 20, exon 21, or exon 22. In some instances, EGFR DNA or RNA comprises one or more mutations in exon 18, exon 19, exon 20, exon 21, exon 22, or a combination thereof.

In some instances, EGFR DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues 34, 38, 45, 62, 63, 77, 78, 108, 114, 120, 140, 148, 149, 160, 177, 178, 189, 191, 198, 220, 222, 223, 229, 237, 240, 244, 252, 254, 255, 256, 263, 270, 273, 276, 282, 288, 289, 301, 303, 304, 309, 314, 326, 331, 354, 363, 373, 337, 380, 384, 393, 427, 428, 437, 441, 447, 465, 475, 515, 526, 527, 531, 536, 541, 546, 571, 588, 589, 596, 596, 598, 602, 614, 620, 628, 636, 641, 645, 651, 671, 689, 694, 700, 709, 712, 714, 715, 716, 719, 720, 721, 731, 733, 739-744, 742, 746-750, 746-752, 746, 747, 747-749, 747-751, 747-753, 751, 752, 754, 752-759, 750, 761-762, 761, 763, 765, 767-768, 767-769, 768, 769, 769-770, 770-771, 772, 773-774, 773, 774, 774-775, 776, 779, 783, 784, 786, 790, 792, 794, 798, 803, 805, 807, 810, 826, 827, 831, 832, 833, 835, 837, 838, 839, 842, 843, 847, 850, 851, 853, 854, 856, 858, 861, 863, 894, 917, 967, 1006, 1019, 1042, 1100, 1129, 1141, 1153, 1164, 1167, or a combination thereof of the EGFR polypeptide. In some embodiments, EGFR DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues 747, 761, 790, 854, 858, or a combination thereof of the EGFR polypeptide. In some embodiments, EGFR DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues 761, 790, 858, or a combination thereof of the EGFR polypeptide. In some embodiments, EGFR DNA or RNA comprises a mutation at a position corresponding to amino acid residue 747 of the EGFR polypeptide. In some embodiments, EGFR DNA or RNA comprises a mutation at a position corresponding to amino acid residue 761 of the EGFR polypeptide. In some embodiments, EGFR DNA or RNA comprises a mutation at a position corresponding to amino acid residue 790 of the EGFR polypeptide. In some embodiments, EGFR DNA or RNA comprises a mutation at a position corresponding to amino acid residue 854 of the EGFR polypeptide. In some embodiments, EGFR DNA or RNA comprises a mutation at a position corresponding to amino acid residue 858 of the EGFR polypeptide.

In some embodiments, EGFR DNA or RNA comprises one or more mutations selected from T34M, L38V, E45Q, L62R, G63R, G63K, S77F, F78L, R108K, R108G, E114K, A120P, L140V, V148M, R149W, E160K, S177P, M178I, K189T, D191N, S198R, S220P, R222L, R222C, S223Y, S229C, A237Y, C240Y, R244G, R252C, R252P, F254I, R255 (nonsense mutation), D256Y, T263P, Y270C, T273A, Q276 (nonsense), E282K, G288 (frame shift), A289D, A289V, A289T, A289N, A289D, V301 (deletion), D303H, H304Y, R309Q, D314N, C326R, G331R, T354M, T363I, P373Q, R337S, S380 (frame shift), T384S, D393Y, R427L, G428S, S437Y, V441I, S447Y, G465R, I475V, C515S, C526S, R527L, R531 (nonsense), V536M, L541I, P546Q, C571S, G588S, P589L, P596L, P596S, P596R, P596L, G598V, G598A, E602G, G614D, C620Y, C620W, C628Y, C628F, C636Y, T638M, P641H, S645C, V651M, R671C, V689M, P694S, N700D, E709A, E709K, E709Q, E709K, F712L, K714N, I715S, K716R, G719A, G719C, G719D, G719S, S720C, S720F, G721V, W731Stop, P733L, K739-I744 (insertion), V742I, V742A, E746-A750 (deletion), E746K, L747S, L747-E749 (deletion), L747-T751 (deletion), L747-P753 (deletion), G746-S752 (deletion), T751I, S752Y, K754 (deletion), S752-I759 (deletion), A750P, D761-E762 (e.g., residues EAFQ (SEQ ID NO: 1893) insertion), D761N, D761Y, A763V, V765A, A767-S768 (e.g., residues TLA insertion), A767-V769 (e.g., residues ASV insertion), S768I, S768T, V769L, V769M, V769-D770 (e.g., residue Y insertion), 770-771 (e.g., residues GL insertion), 770-771 (e.g., residue G insertion), 770-771 (e.g., residues CV insertion), 770-771 (e.g., residues SVD insertion), P772R, 773-774 (e.g., residues NPH insertion), H773R, H773L, V774M, 774-775 (e.g., residues HV insertion), R776H, R776C, G779F, T783A, T784F, T854A, V786L, T790M, L792P, P794H, L798F, R803W, H805R, D807H, G810S, N826S, Y827 (nonsense), R831H, R832C, R832H, L833F, L833V, H835L, D837V, L838M, L838P, A839V, N842H, V843L, T847K, T847I, H850N, V851A, I853T, F856L, L858R, L858M, L861Q, L861R, G863D, Q894L, G917A, E967A, D1006Y, P1019L, S1042N, R1100S, H1129Y, T1141S, S1153I, Q1164R, L1167M, or a combination thereof of the EGFR polypeptide.

In some instances, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising one or more mutations. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising one or more mutations in exon 18, exon 19, exon 20, exon 21, exon 22, or a combination thereof.

In some embodiments, a polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising one or more mutations at positions corresponding to amino acid residues 34, 38, 45, 62, 63, 77, 78, 108, 114, 120, 140, 148, 149, 160, 177, 178, 189, 191, 198, 220, 222, 223, 229, 237, 240, 244, 252, 254, 255, 256, 263, 270, 273, 276, 282, 288, 289, 301, 303, 304, 309, 314, 326, 331, 354, 363, 373, 337, 380, 384, 393, 427, 428, 437, 441, 447, 465, 475, 515, 526, 527, 531, 536, 541, 546, 571, 588, 589, 596, 596, 598, 602, 614, 620, 628, 636, 641, 645, 651, 671, 689, 694, 700, 709, 712, 714, 715, 716, 719, 720, 721, 731, 733, 739-744, 742, 746-750, 746-752, 746, 747, 747-749, 747-751, 747-753, 751, 752, 754, 752-759, 750, 761-762, 761, 763, 765, 767-768, 767-769, 768, 769, 769-770, 770-771, 772, 773-774, 773, 774, 774-775, 776, 779, 783, 784, 786, 790, 792, 794, 798, 803, 805, 807, 810, 826, 827, 831, 832, 833, 835, 837, 838, 839, 842, 843, 847, 850, 851, 853, 854, 856, 858, 861, 863, 894, 917, 967, 1006, 1019, 1042, 1100, 1129, 1141, 1153, 1164, 1167, or a combination thereof of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising one or more mutations at positions corresponding to amino acid residues 747, 761, 790, 854, 858, or a combination thereof of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising one or more mutations at positions corresponding to amino acid residues 761, 790, 858, or a combination thereof of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising a mutation at a position corresponding to amino acid residue 747 of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising a mutation at a position corresponding to amino acid residue 761 of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising a mutation at a position corresponding to amino acid residue 790 of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising a mutation at a position corresponding to amino acid residue 854 of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising a mutation at a position corresponding to amino acid residue 858 of the EGFR polypeptide.

In some embodiments, a polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising one or more mutations selected from T34M, L38V, E45Q, L62R, G63R, G63K, S77F, F78L, R108K, R108G, E114K, A120P, L140V, V148M, R149W, E160K, S177P, M178I, K189T, D191N, S198R, S220P, R222L, R222C, S223Y, S229C, A237Y, C240Y, R244G, R252C, R252P, F254I, R255 (nonsense mutation), D256Y, T263P, Y270C, T273A, Q276 (nonsense), E282K, G288 (frame shift), A289D, A289V, A289T, A289N, A289D, V301 (deletion), D303H, H304Y, R309Q, D314N, C326R, G331R, T354M, T363I, P373Q, R337S, S380 (frame shift), T384S, D393Y, R427L, G428S, S437Y, V441I, S447Y, G465R, I475V, C515S, C526S, R527L, R531 (nonsense), V536M, L541I, P546Q, C571S, G588S, P589L, P596L, P596S, P596R, P596L, G598V, G598A, E602G, G614D, C620Y, C620W, C628Y, C628F, C636Y, T638M, P641H, S645C, V651M, R671C, V689M, P694S, N700D, E709A, E709K, E709Q, E709K, F712L, K714N, I715S, K716R, G719A, G719C, G719D, G719S, S720C, S720F, G721V, W731Stop, P733L, K739-I744 (insertion), V742I, V742A, E746-A750 (deletion), E746K, L747S, L747-E749 (deletion), L747-T751 (deletion), L747-P753 (deletion), G746-S752 (deletion), T751I, S752Y, K754 (deletion), S752-I759 (deletion), A750P, D761-E762 (e.g., residues EAFQ (SEQ ID NO: 1893) insertion), D761N, D761Y, A763V, V765A, A767-S768 (e.g., residues TLA insertion), A767-V769 (e.g., residues ASV insertion), S768I, S768T, V769L, V769M, V769-D770 (e.g., residue Y insertion), 770-771 (e.g., residues GL insertion), 770-771 (e.g., residue G insertion), 770-771 (e.g., residues CV insertion), 770-771 (e.g., residues SVD insertion), P772R, 773-774 (e.g., residues NPH insertion), H773R, H773L, V774M, 774-775 (e.g., residues HV insertion), R776H, R776C, G779F, T783A, T784F, T854A, V786L, T790M, L792P, P794H, L798F, R803W, H805R, D807H, G810S, N826S, Y827 (nonsense), R831H, R832C, R832H, L833F, L833V, H835L, D837V, L838M, L838P, A839V, N842H, V843L, T847K, T847I, H850N, V851A, I853T, F856L, L858R, L858M, L861Q, L861R, G863D, Q894L, G917A, E967A, D1006Y, P1019L, S1042N, R1100S, H1129Y, T1141S, S1153I, Q1164R, L1167M, or a combination thereof of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising one or more mutations selected from L747S, D761Y, T790M, T854A, L858R, or a combination thereof of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising one or more mutations selected from D761Y, T790M, L858R, or a combination thereof of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising mutation L747S of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising mutation D761Y of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising mutation T790M of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising mutation T854A of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA comprising mutation L858R of the EGFR polypeptide.

In some embodiments, a polynucleic acid molecule comprises a sequence that hybridizes to a target sequence illustrated in Table 1. In some embodiments, the polynucleic acid molecule hybridizes to an EGFR target sequence selected from SEQ ID NOs: 1-376. In some cases, the polynucleic acid molecule hybridizes to an EGFR target sequence selected from SEQ ID NOs: 1-376 with less than 5 mismatched bases, with less than 4 mismatched bases, with less than 3 mismatched bases, with less than 2 mismatched bases, or with 1 mismatched base. In some cases, the polynucleic acid molecule hybridizes to an EGFR target sequence selected from SEQ ID NOs: 1-376 with less than 4 mismatched bases.

In some embodiments, a polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listed in Table 2, Table 3, or Table 6. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 377-1892. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NOs: 377-1892. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NOs: 377-1892. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NOs: 377-1892. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NOs: 377-1892. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NOs: 377-1892. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NOs: 377-1892. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NOs: 377-1892. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NOs: 377-1892. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NOs: 377-1892. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NOs: 377-1892. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NOs: 377-1892. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NOs: 377-1892. In some embodiments, the polynucleic acid molecule consists of SEQ ID NOs: 377-1892.

In some embodiments, a polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some instances, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 377-1892. In some cases, the second polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 377-1892. In some cases, the polynucleic acid molecule comprises a first polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 377-1892 and a second polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 377-1892.

In some embodiments, a polynucleic acid molecule described herein comprises RNA or DNA. In some cases, the polynucleic acid molecule comprises RNA. In some instances, RNA comprises short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heterogeneous nuclear RNA (hnRNA). In some instances, RNA comprises shRNA. In some instances, RNA comprises miRNA. In some instances, RNA comprises dsRNA. In some instances, RNA comprises tRNA. In some instances, RNA comprises rRNA. In some instances, RNA comprises hnRNA. In some instances, the RNA comprises siRNA. In some instances, the polynucleic acid molecule comprises siRNA.

In some embodiments, a polynucleic acid molecule described herein is from about 10 to about 50 nucleotides in length. In some instances, the polynucleic acid molecule is from about 10 to about 30, from about 15 to about 30, from about 18 to about 25, from about 18 to about 24, from about 19 to about 23, or from about 20 to about 22 nucleotides in length.

In some embodiments, a polynucleic acid molecule is about 50 nucleotides in length. In some instances, the polynucleic acid molecule is about 45 nucleotides in length. In some instances, the polynucleic acid molecule is about 40 nucleotides in length. In some instances, the polynucleic acid molecule is about 35 nucleotides in length. In some instances, the polynucleic acid molecule is about 30 nucleotides in length. In some instances, the polynucleic acid molecule is about 25 nucleotides in length. In some instances, the polynucleic acid molecule is about 20 nucleotides in length. In some instances, the polynucleic acid molecule is about 19 nucleotides in length. In some instances, the polynucleic acid molecule is about 18 nucleotides in length. In some instances, the polynucleic acid molecule is about 17 nucleotides in length. In some instances, the polynucleic acid molecule is about 16 nucleotides in length. In some instances, the polynucleic acid molecule is about 15 nucleotides in length. In some instances, the polynucleic acid molecule is about 14 nucleotides in length. In some instances, the polynucleic acid molecule is about 13 nucleotides in length. In some instances, the polynucleic acid molecule is about 12 nucleotides in length. In some instances, the polynucleic acid molecule is about 11 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 nucleotides in length. In some instances, the polynucleic acid molecule is between about 10 and about 50 nucleotides in length. In some instances, the polynucleic acid molecule is from about 10 to about 45 nucleotides in length. In some instances, the polynucleic acid molecule is from about 10 to about 40 nucleotides in length. In some instances, the polynucleic acid molecule is from about 10 to about 35 nucleotides in length. In some instances, the polynucleic acid molecule is from about 10 to about 30 nucleotides in length. In some instances, the polynucleic acid molecule is from about 10 to about 25 nucleotides in length. In some instances, the polynucleic acid molecule is from about 10 to about 20 nucleotides in length. In some instances, the polynucleic acid molecule is from about 15 to about 25 nucleotides in length. In some instances, the polynucleic acid molecule is from about 15 to about 30 nucleotides in length. In some instances, the polynucleic acid molecule is from about 12 to about 30 nucleotides in length.

In some embodiments, a polynucleic acid molecule described herein comprises a first polynucleotide. In some instances, the polynucleic acid molecule comprises a second polynucleotide. In some instances, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some instances, the first polynucleotide is a sense strand or passenger strand. In some instances, the second polynucleotide is an antisense strand or guide strand.

In some embodiments, a polynucleic acid molecule is a first polynucleotide. In some embodiments, the first polynucleotide is from about 10 to about 50 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 30, from about 15 to about 30, from about 18 to about 25, from about 18 to about 24, from about 19 to about 23, or from about 20 to about 22 nucleotides in length.

In some instances, a first polynucleotide is about 50 nucleotides in length. In some instances, the first polynucleotide is about 45 nucleotides in length. In some instances, the first polynucleotide is about 40 nucleotides in length. In some instances, the first polynucleotide is about 35 nucleotides in length. In some instances, the first polynucleotide is about 30 nucleotides in length. In some instances, the first polynucleotide is about 25 nucleotides in length. In some instances, the first polynucleotide is about 20 nucleotides in length. In some instances, the first polynucleotide is about 19 nucleotides in length. In some instances, the first polynucleotide is about 18 nucleotides in length. In some instances, the first polynucleotide is about 17 nucleotides in length. In some instances, the first polynucleotide is about 16 nucleotides in length. In some instances, the first polynucleotide is about 15 nucleotides in length. In some instances, the first polynucleotide is about 14 nucleotides in length. In some instances, the first polynucleotide is about 13 nucleotides in length. In some instances, the first polynucleotide is about 12 nucleotides in length. In some instances, the first polynucleotide is about 11 nucleotides in length. In some instances, the first polynucleotide is about 10 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 50 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 45 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 40 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 35 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 30 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 25 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 20 nucleotides in length. In some instances, the first polynucleotide is from about 15 to about 25 nucleotides in length. In some instances, the first polynucleotide is from about 15 to about 30 nucleotides in length. In some instances, the first polynucleotide is from about 12 to about 30 nucleotides in length.

In some embodiments, a polynucleic acid molecule is a second polynucleotide. In some embodiments, the second polynucleotide is from about 10 to about 50 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 30, from about 15 to about 30, from about 18 to about 25, from about 18 to about 24, from about 19 to about 23, or from about 20 to about 22 nucleotides in length.

In some instances, a second polynucleotide is about 50 nucleotides in length. In some instances, the second polynucleotide is about 45 nucleotides in length. In some instances, the second polynucleotide is about 40 nucleotides in length. In some instances, the second polynucleotide is about 35 nucleotides in length. In some instances, the second polynucleotide is about 30 nucleotides in length. In some instances, the second polynucleotide is about 25 nucleotides in length. In some instances, the second polynucleotide is about 20 nucleotides in length. In some instances, the second polynucleotide is about 19 nucleotides in length. In some instances, the second polynucleotide is about 18 nucleotides in length. In some instances, the second polynucleotide is about 17 nucleotides in length. In some instances, the second polynucleotide is about 16 nucleotides in length. In some instances, the second polynucleotide is about 15 nucleotides in length. In some instances, the second polynucleotide is about 14 nucleotides in length. In some instances, the second polynucleotide is about 13 nucleotides in length. In some instances, the second polynucleotide is about 12 nucleotides in length. In some instances, the second polynucleotide is about 11 nucleotides in length. In some instances, the second polynucleotide is about 10 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 50 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 45 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 40 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 35 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 30 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 25 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 20 nucleotides in length. In some instances, the second polynucleotide is from about 15 to about 25 nucleotides in length. In some instances, the second polynucleotide is from about 15 to about 30 nucleotides in length. In some instances, the second polynucleotide is from about 12 to about 30 nucleotides in length.

In some embodiments, a polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some instances, the polynucleic acid molecule further comprises a blunt terminus, an overhang, or a combination thereof. In some instances, the blunt terminus is a 5′ blunt terminus, a 3′ blunt terminus, or both. In some cases, the overhang is a 5′ overhang, 3′ overhang, or both. In some cases, the overhang comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-base pairing nucleotides. In some cases, the overhang comprises 1, 2, 3, 4, 5, or 6 non-base pairing nucleotides. In some cases, the overhang comprises 1, 2, 3, or 4 non-base pairing nucleotides. In some cases, the overhang comprises 1 non-base pairing nucleotide. In some cases, the overhang comprises 2 non-base pairing nucleotides. In some cases, the overhang comprises 3 non-base pairing nucleotides. In some cases, the overhang comprises 4 non-base pairing nucleotides.

In some embodiments, the sequence of a polynucleic acid molecule is at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 50% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 60% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 70% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 80% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 90% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 95% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 99% complementary to a target sequence described herein. In some instances, the sequence of the polynucleic acid molecule is 100% complementary to a target sequence described herein.

In some embodiments, the sequence of a polynucleic acid molecule has 5 or less mismatches to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule has 4 or less mismatches to a target sequence described herein. In some instances, the sequence of the polynucleic acid molecule has 3 or less mismatches to a target sequence described herein. In some cases, the sequence of the polynucleic acid molecule has 2 or less mismatches to a target sequence described herein. In some cases, the sequence of the polynucleic acid molecule has 1 or less mismatches to a target sequence described herein.

In some embodiments, the specificity of a polynucleic acid molecule that hybridizes to a target sequence described herein is a 95%, 98%, 99%, 99.5% or 100% sequence complementarity of the polynucleic acid molecule to a target sequence. In some instances, the hybridization is a high stringent hybridization condition.

In some embodiments, the polynucleic acid molecule hybridizes to at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 8 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 9 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 10 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 11 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 12 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 15 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 18 contiguous bases of a target sequence described herein.

In some embodiments, a polynucleic acid molecule has reduced off-target effect. In some instances, “off-target” or “off-target effects” refer to any instance in which a polynucleic acid polymer directed against a given target causes an unintended effect by interacting either directly or indirectly with another mRNA sequence, a DNA sequence or a cellular protein or other moiety. In some instances, an “off-target effect” occurs when there is a simultaneous degradation of other transcripts due to partial homology or complementarity between that other transcript and the sense and/or antisense strand of the polynucleic acid molecule.

In some embodiments, a polynucleic acid molecule comprises natural, synthetic, or artificial nucleotide analogues or bases. In some cases, the polynucleic acid molecule comprises combinations of DNA, RNA and/or nucleotide analogues. In some instances, the synthetic or artificial nucleotide analogues or bases comprise modifications at one or more of ribose moiety, phosphate moiety, nucleoside moiety, or a combination thereof.

In some embodiments, nucleotide analogues or artificial nucleotide base comprise a nucleic acid with a modification at a 2′ hydroxyl group of the ribose moiety. In some instances, the modification includes an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety. Exemplary alkyl moiety includes, but is not limited to, halogens, sulfurs, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols and oxygen. In some instances, the alkyl moiety further comprises a modification. In some instances, the modification comprises an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso, group, a nitrile group, a heterocycle (e.g., imidazole, hydrazino or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some instances, the alkyl moiety further comprises a hetero substitution. In some instances, the carbon of the heterocyclic group is substituted by a nitrogen, oxygen or sulfur. In some instances, the heterocyclic substitution includes but is not limited to, morpholino, imidazole, and pyrrolidino.

In some instances, the modification at the 2′ hydroxyl group is a 2′-O-methyl modification or a 2′-O-methoxyethyl (2′-O-MOE) modification. In some cases, the 2′-O-methyl modification adds a methyl group to the 2′ hydroxyl group of the ribose moiety whereas the 2′O-methoxyethyl modification adds a methoxyethyl group to the 2′ hydroxyl group of the ribose moiety. Exemplary chemical structures of a 2′-O-methyl modification of an adenosine molecule and 2′O-methoxyethyl modification of a uridine are illustrated below.

In some instances, the modification at the 2′ hydroxyl group is a 2′-O-aminopropyl modification in which an extended amine group comprising a propyl linker binds the amine group to the 2′ oxygen. In some instances, this modification neutralizes the phosphate-derived overall negative charge of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar and thereby improves cellular uptake properties due to its zwitterionic properties. An exemplary chemical structure of a 2′-O-aminopropyl nucleoside phosphoramidite is illustrated below.

In some instances, the modification at the 2′ hydroxyl group is a locked or bridged ribose modification (e.g., locked nucleic acid or LNA) in which the oxygen molecule bound at the 2′ carbon is linked to the 4′ carbon by a methylene group, thus forming a 2′-C,4′-C-oxy-methylene-linked bicyclic ribonucleotide monomer. Exemplary representations of the chemical structure of LNA are illustrated below. The representation shown to the left highlights the chemical connectivities of an LNA monomer. The representation shown to the right highlights the locked 3′-endo (³E) conformation of the furanose ring of an LNA monomer.

In some instances, the modification at the 2′ hydroxyl group comprises ethylene nucleic acids (ENA) such as for example 2′-4′-ethylene-bridged nucleic acid, which locks the sugar conformation into a C₃′-endo sugar puckering conformation. ENA are part of the bridged nucleic acids class of modified nucleic acids that also comprises LNA. Exemplary chemical structures of the ENA and bridged nucleic acids are illustrated below.

In some embodiments, additional modifications at the 2′ hydroxyl group include 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA).

In some embodiments, nucleotide analogues comprise modified bases such as, but not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5-(2-amino) propyl uridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2, 2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides (such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, or 6-azothymidine), 5-methyl-2-thiouridine, other thio bases (such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl groups, any O-and N-alkylated purines and pyrimidines (such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, or pyridine-2-one), phenyl and modified phenyl groups (such as aminophenol or 2,4,6-trimethoxy benzene), modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyi nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moieties, in some cases, are or are based on mannoses, arabinoses, glucopyranoses, galactopyranoses, 4′-thioribose, and other sugars, heterocycles, or carbocycles. The term nucleotide also includes what are known in the art as universal bases. By way of example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine.

In some embodiments, nucleotide analogues further comprise morpholinos, peptide nucleic acids (PNAs), methylphosphonate nucleotides, thiolphosphonate nucleotides, 2′-fluoro N3-P5′-phosphoramidites, 1′,5′-anhydrohexitol nucleic acids (HNAs), or a combination thereof. Morpholino or phosphorodiamidate morpholino oligo (PMO) comprises synthetic molecules whose structure mimics natural nucleic acid structure but deviates from the normal sugar and phosphate structures. In some instances, the five member ribose ring is substituted with a six member morpholino ring containing four carbons, one nitrogen, and one oxygen. In some cases, the ribose monomers are linked by a phosphordiamidate group instead of a phosphate group. In such cases, the backbone alterations remove all positive and negative charges making morpholinos neutral molecules capable of crossing cellular membranes without the aid of cellular delivery agents such as those used by charged oligonucleotides.

In some embodiments, peptide nucleic acid (PNA) does not contain sugar ring or phosphate linkage and the bases are attached and appropriately spaced by oligoglycine-like molecules, therefore eliminating a backbone charge.

In some embodiments, one or more modifications optionally occur at the internucleotide linkage. In some instances, modified internucleotide linkage includes, but is not limited to, phosphorothioates; phosphorodithioates; methylphosphonates; 5′-alkylenephosphonates; 5′-methylphosphonate; 3′-alkylene phosphonates; borontrifluoridates; borano phosphate esters and selenophosphates of 3′-5′linkage or 2′-5′linkage; phosphotriesters; thionoalkylphosphotriesters; hydrogen phosphonate linkages; alkyl phosphonates; alkylphosphonothioates; arylphosphonothioates; phosphoroselenoates; phosphorodiselenoates; phosphinates; phosphoramidates; 3′-alkylphosphoramidates; aminoalkylphosphoramidates; thionophosphoramidates; phosphoropiperazidates; phosphoroanilothioates; phosphoroanilidates; ketones; sulfones; sulfonamides; carbonates; carbamates; methylenehydrazos; methylenedimethylhydrazos; formacetals; thioformacetals; oximes; methyleneiminos; methylenemethyliminos; thioamidates; linkages with riboacetyl groups; aminoethyl glycine; silyl or siloxane linkages; alkyl or cycloalkyl linkages with or without heteroatoms of, for example, 1 to 10 carbons that are saturated or unsaturated and/or substituted and/or contain heteroatoms; linkages with morpholino structures, amides, or polyamides wherein the bases are attached to the aza nitrogens of the backbone directly or indirectly; and combinations thereof.

In some instances, the modification is a methyl or thiol modification such as methylphosphonate or thiolphosphonate modification. Exemplary thiolphosphonate nucleotide (left) and methylphosphonate nucleotide (right) are illustrated below.

In some instances, a modified nucleotide includes, but is not limited to, 2′-fluoro N3-P5′-phosphoramidites illustrated as:

In some instances, a modified nucleotide includes, but is not limited to, hexitol nucleic acid (or 1′,5′-anhydrohexitol nucleic acids (HNA)) illustrated as:

In some embodiments, one or more modifications further optionally include modifications of the ribose moiety, phosphate backbone and the nucleoside, or modifications of the nucleotide analogues at the 3′ or the 5′ terminus. For example, the 3′ terminus optionally include a 3′ cationic group, or by inverting the nucleoside at the 3′-terminus with a 3′-3′ linkage. In another alternative, the 3′-terminus is optionally conjugated with an aminoalkyl group, e.g., a 3′ C5-aminoalkyl dT. In an additional alternative, the 3′-terminus is optionally conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site. In some instances, the 5′-terminus is conjugated with an aminoalkyl group, e.g., a 5′-O-alkylamino substituent. In some cases, the 5′-terminus is conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site.

In some embodiments, a polynucleic acid molecule comprises one or more artificial nucleotide analogues described herein. In some instances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more artificial nucleotide analogues described herein. In some embodiments, the artificial nucleotide analogues include 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2′-fluoro N3-P5′-phosphoramidites, or a combination thereof. In some instances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of the artificial nucleotide analogues selected from 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2′-fluoro N3-P5′-phosphoramidites, or a combination thereof. In some instances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of 2′-O-methyl modified nucleotides. In some instances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of 2′-O-methoxyethyl (2′-O-MOE) modified nucleotides. In some instances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of thiolphosphonate nucleotides.

In some instances, a polynucleic acid molecule comprises at least one of: from about 5% to about 100% modification, from about 10% to about 100% modification, from about 20% to about 100% modification, from about 30% to about 100% modification, from about 40% to about 100% modification, from about 50% to about 100% modification, from about 60% to about 100% modification, from about 70% to about 100% modification, from about 80% to about 100% modification, and from about 90% to about 100% modification. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 377-1128.

In some cases, a polynucleic acid molecule comprises at least one of: from about 10% to about 90% modification, from about 20% to about 90% modification, from about 30% to about 90% modification, from about 40% to about 90% modification, from about 50% to about 90% modification, from about 60% to about 90% modification, from about 70% to about 90% modification, and from about 80% to about 100% modification. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 377-1128.

In some cases, a polynucleic acid molecule comprises at least one of: from about 10% to about 80% modification, from about 20% to about 80% modification, from about 30% to about 80% modification, from about 40% to about 80% modification, from about 50% to about 80% modification, from about 60% to about 80% modification, and from about 70% to about 80% modification. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 377-1128.

In some instances, a polynucleic acid molecule comprises at least one of: from about 10% to about 70% modification, from about 20% to about 70% modification, from about 30% to about 70% modification, from about 40% to about 70% modification, from about 50% to about 70% modification, and from about 60% to about 70% modification. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 377-1128.

In some instances, a polynucleic acid molecule comprises at least one of: from about 10% to about 60% modification, from about 20% to about 60% modification, from about 30% to about 60% modification, from about 40% to about 60% modification, and from about 50% to about 60% modification. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 377-1128.

In some cases, a polynucleic acid molecule comprises at least one of: from about 10% to about 50% modification, from about 20% to about 50% modification, from about 30% to about 50% modification, and from about 40% to about 50% modification. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 377-1128.

In some cases, a polynucleic acid molecule comprises at least one of: from about 10% to about 40% modification, from about 20% to about 40% modification, and from about 30% to about 40% modification. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 377-1128.

In some cases, a polynucleic acid molecule comprises at least one of: from about 10% to about 30% modification, and from about 20% to about 30% modification. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 377-1128.

In some cases, a polynucleic acid molecule comprises from about 10% to about 20% modification. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 377-1128.

In some cases, a polynucleic acid molecule comprises from about 15% to about 90%, from about 20% to about 80%, from about 30% to about 70%, or from about 40% to about 60% modifications. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 377-1128.

In additional cases, a polynucleic acid molecule comprises at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modification. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 377-1128.

In some embodiments, a polynucleic acid molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, or more modifications. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 377-1128.

In some instances, a polynucleic acid molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, or more modified nucleotides. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 377-1128.

In some instances, from about 5 to about 100% of a polynucleic acid molecule comprise an artificial nucleotide analogue described herein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the polynucleic acid molecule comprise an artificial nucleotide analogue described herein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a polynucleic acid molecule of SEQ ID NOs: 377-1892 comprise an artificial nucleotide analogue described herein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 5% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 10% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 15% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 20% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 25% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 30% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 35% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 40% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 45% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 50% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 55% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 60% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 65% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 70% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 75% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 80% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 85% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 90% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 95% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 96% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 97% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 98% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 99% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some instances, about 100% of a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprise an artificial nucleotide analogue described herein. In some embodiments, the artificial nucleotide analogue comprises 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2′-fluoro N3-P5′-phosphoramidites, or a combination thereof.

In some embodiments, a polynucleic acid molecule comprises from about 1 to about 25 modifications in which the modification comprises an artificial nucleotide analogues described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises from about 1 to about 25 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 1 modification in which the modification comprises an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 2 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 3 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 4 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 5 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 6 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 7 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 8 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 9 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 10 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 11 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 12 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 13 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 14 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 15 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 16 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 17 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 18 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 19 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 20 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 21 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 22 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 23 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 24 modifications in which the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, a polynucleic acid molecule of SEQ ID NOs: 377-1128 comprises about 25 modifications in which the modifications comprise an artificial nucleotide analogue described herein.

In some instances, a polynucleic acid molecule that comprises an artificial nucleotide analogue comprises a sequence selected from SEQ ID NOs: 1129-1892.

In some embodiments, a polynucleic acid molecule is assembled from two separate polynucleotides wherein one polynucleotide comprises the sense strand and the second polynucleotide comprises the antisense strand of the polynucleic acid molecule. In other embodiments, the sense strand is connected to the antisense strand via a linker molecule, which in some instances, is a polynucleotide linker or a non-nucleotide linker.

In some embodiments, a polynucleic acid molecule comprises a sense strand and antisense strand, wherein pyrimidine nucleotides in the sense strand comprise 2′-O-methylpyrimidine nucleotides and purine nucleotides in the sense strand comprise 2′-deoxy purine nucleotides. In some embodiments, a polynucleic acid molecule comprises a sense strand and antisense strand, wherein pyrimidine nucleotides present in the sense strand comprise 2′-deoxy-2′-fluoro pyrimidine nucleotides and wherein purine nucleotides present in the sense strand comprise 2′-deoxy purine nucleotides.

In some embodiments, a polynucleic acid molecule comprises a sense strand and antisense strand, wherein the pyrimidine nucleotides when present in said antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides when present in said antisense strand are 2′-O-methyl purine nucleotides.

In some embodiments, a polynucleic acid molecule comprises a sense strand and antisense strand, wherein the pyrimidine nucleotides when present in said antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and wherein the purine nucleotides when present in said antisense strand comprise 2′-deoxy-purine nucleotides.

In some embodiments, a polynucleic acid molecule comprises a sense strand and antisense strand, wherein the sense strand includes a terminal cap moiety at the 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the sense strand. In other embodiments, the terminal cap moiety is an inverted deoxy abasic moiety.

In some embodiments, a polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises a phosphate backbone modification at the 3′ end of the antisense strand. In some instances, the phosphate backbone modification is a phosphorothioate.

In some embodiments, a polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises a glyceryl modification at the 3′ end of the antisense strand.

In some embodiments, a polynucleic acid molecule comprises a sense strand and an antisense strand, in which the sense strand comprises one or more (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and in which the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In other embodiments, one or more (for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine nucleotides of the sense and/or antisense strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more (for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In some embodiments, a polynucleic acid molecule comprises a sense strand and an antisense strand, in which the sense strand comprises about 1 to about 25 (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and in which the antisense strand comprises about 1 to about 25 or more (for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In other embodiments, one or more (for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine nucleotides of the sense and/or antisense strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 25 or more (for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In some embodiments, a polynucleic acid molecule comprises a sense strand and an antisense strand, in which the antisense strand comprises one or more (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In other embodiments, one or more (for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) pyrimidine nucleotides of the sense and/or antisense strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends, being present in the same or different strand.

In some embodiments, a polynucleic acid molecule comprises a sense strand and an antisense strand, in which the antisense strand comprises about 1 to about 25 or more (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 25 or more (for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In other embodiments, one or more (for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) pyrimidine nucleotides of the sense and/or antisense strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5 (for example about 1, 2, 3, 4, 5 or more) phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In some embodiments, a polynucleic acid molecule described herein is a chemically-modified short interfering nucleic acid molecule having about 1 to about 25 (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) phosphorothioate internucleotide linkages in each strand of the polynucleic acid molecule.

In another embodiment, a polynucleic acid molecule described herein comprises 2′-5′ internucleotide linkages. In some instances, the 2′-5′ internucleotide linkage(s) is at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of one or both sequence strands. In addition instances, the 2′-5′ internucleotide linkage(s) is present at various other positions within one or both sequence strands (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) including every internucleotide linkage of a pyrimidine nucleotide in one or both strands of the polynucleic acid molecule comprise a 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, including every internucleotide linkage of a purine nucleotide in one or both strands of the polynucleic acid molecule comprise a 2′-5′ internucleotide linkage.

In some embodiments, a polynucleic acid molecule is a single stranded polynucleic acid molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the polynucleic acid molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the polynucleic acid are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein one or more purine nucleotides present in the polynucleic acid are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides), and a terminal cap modification, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the polynucleic acid molecule optionally further comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2′-deoxynucleotides at the 3′-end of the polynucleic acid molecule, wherein the terminal nucleotides further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages, and wherein the polynucleic acid molecule optionally further comprises a terminal phosphate group, such as a 5′-terminal phosphate group.

In some cases, one or more artificial nucleotide analogues described herein are resistant toward nucleases such as for example ribonuclease such as RNase H, deoxyribunuclease such as DNase, or exonuclease such as 5′-3′ exonuclease and 3′-5′ exonuclease, when compared to natural polynucleic acid molecules. In some instances, artificial nucleotide analogues comprising 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2′-fluoro N3-P5′-phosphoramidites, or combinations thereof are resistant toward nucleases such as for example ribonuclease such as RNase H, deoxyribunuclease such as DNase, or exonuclease such as 5′-3′ exonuclease and 3′-5′ exonuclease. In some instances, 2′-O-methyl modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, 2′O-methoxyethyl (2′-O-MOE) modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, 2′-O-aminopropyl modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, 2′-deoxy modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, T-deoxy-2′-fluoro modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, 2′-O-aminopropyl (2′-O-AP) modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, 2′-O-dimethylaminoethyl (2′-O-DMAOE) modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, 2′-O-dimethylaminopropyl (2′-O-DMAP) modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE) modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, 2′-O—N-methylacetamido (2′-O-NMA) modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, LNA modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, ENA modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, HNA modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, morpholinos is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, PNA modified polynucleic acid molecule is resistant to nucleases (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, methylphosphonate nucleotides modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, thiolphosphonate nucleotides modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, polynucleic acid molecule comprising 2′-fluoro N3-P5′-phosphoramidites is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances, the 5′ conjugates described herein inhibit 5′-3′ exonucleolytic cleavage. In some instances, the 3′ conjugates described herein inhibit exonucleolytic cleavage.

In some embodiments, one or more of the artificial nucleotide analogues described herein have increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. The one or more of the artificial nucleotide analogues comprising 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, or 2′-fluoro N3-P5′-phosphoramidites have increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2′-O-methyl-modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2′-O-methoxyethyl (2′-O-MOE) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2′-O-aminopropyl modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2′-deoxy modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, T-deoxy-2′-fluoro modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2′-O-aminopropyl (2′-O-AP) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2′-O-dimethylaminoethyl (2′-O-DMAOE) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2′-O-dimethylaminopropyl (2′-O-DMAP) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2′-O—N-methylacetamido (2′-O-NMA) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, LNA-modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, ENA-modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, PNA-modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, HNA-modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, morpholino-modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, methylphosphonate nucleotide-modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, thiolphosphonate nucleotide-modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, polynucleic acid molecule comprising 2′-fluoro N3-P5′-phosphoramidites has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some cases, the increased affinity is illustrated with a lower Kd, a higher melt temperature (Tm), or a combination thereof.

In some embodiments, a polynucleic acid molecule described herein is a chirally pure (or stereo pure) polynucleic acid molecule, or a polynucleic acid molecule comprising a single enantiomer. In some instances, the polynucleic acid molecule comprises L-nucleotide. In some instances, the polynucleic acid molecule comprises D-nucleotides. In some instance, a polynucleic acid molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its mirror enantiomer. In some cases, a polynucleic acid molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a racemic mixture. In some instances, the polynucleic acid molecule is a polynucleic acid molecule described in: U.S. Patent Publication Nos: 2014/194610 and 2015/211006; and PCT Publication No.: WO2015107425.

In some embodiments, a polynucleic acid molecule described herein is further modified to include an aptamer-conjugating moiety. In some instances, the aptamer conjugating moiety is a DNA aptamer-conjugating moiety. In some instances, the aptamer-conjugating moiety is Alphamer (Centauri Therapeutics), which comprises an aptamer portion that recognizes a specific cell-surface target and a portion that presents a specific epitopes for attaching to circulating antibodies. In some instance, a polynucleic acid molecule described herein is further modified to include an aptamer-conjugating moiety as described in: U.S. Pat. Nos. 8,604,184, 8,591,910, and 7,850,975.

In additional embodiments, a polynucleic acid molecule described herein is modified to increase its stability. In some embodiment, the polynucleic acid molecule is RNA (e.g., siRNA), and the polynucleic acid molecule is modified to increase its stability. In some instances, the polynucleic acid molecule is modified by one or more of the modifications described above to increase its stability. In some cases, the polynucleic acid molecule is modified at the 2′ hydroxyl position, such as by 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modification or by a locked or bridged ribose conformation (e.g., LNA or ENA). In some cases, the polynucleic acid molecule is modified by 2′-O-methyl and/or 2′-O-methoxyethyl ribose. In some cases, the polynucleic acid molecule also includes morpholinos, PNAs, HNA, methylphosphonate nucleotides, thiolphosphonate nucleotides, and/or 2′-fluoro N3-P5′-phosphoramidites to increase its stability. In some instances, the polynucleic acid molecule is a chirally pure (or stereo pure) polynucleic acid molecule. In some instances, the chirally pure (or stereo pure) polynucleic acid molecule is modified to increase its stability. Suitable modifications to the RNA to increase stability for delivery will be apparent to the skilled person.

In some embodiments, a polynucleic acid molecule describe herein has RNAi activity that modulates expression of RNA encoded by EGFR. In some instances, a polynucleic acid molecule described herein is a double-stranded siRNA molecule that down-regulates expression of EGFR, wherein one of the strands of the double-stranded siRNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of EGFR or RNA encoded by EGFR or a portion thereof, and wherein the second strand of the double-stranded siRNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of EGFR or RNA encoded by EGFR or a portion thereof. In some cases, a polynucleic acid molecule described herein is a double-stranded siRNA molecule that down-regulates expression of EGFR, wherein each strand of the siRNA molecule comprises about 15 to 25, 18 to 24, or 19 to about 23 nucleotides, and wherein each strand comprises at least about 14, 17, or 19 nucleotides that are complementary to the nucleotides of the other strand. In some cases, a polynucleic acid molecule described herein is a double-stranded siRNA molecule that down-regulates expression of EGFR, wherein each strand of the siRNA molecule comprises about 19 to about 23 nucleotides, and wherein each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand. In some instances, the RNAi activity occurs within a cell. In other instances, the RNAi activity occurs in a reconstituted in vitro system.

In some embodiments, a polynucleic acid molecule describe herein has RNAi activity that modulates expression of RNA encoded by EGFR. In some instances, a polynucleic acid molecule described herein is a single-stranded siRNA molecule that down-regulates expression of EGFR, wherein the single-stranded siRNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of EGFR or RNA encoded by EGFR or a portion thereof. In some cases, a polynucleic acid molecule described herein is a single-stranded siRNA molecule that down-regulates expression of EGFR, wherein the siRNA molecule comprises about 15 to 25, 18 to 24, or 19 to about 23 nucleotides. In some cases, a polynucleic acid molecule described herein is a single-stranded siRNA molecule that down-regulates expression of EGFR, wherein the siRNA molecule comprises about 19 to about 23 nucleotides. In some instances, the RNAi activity occurs within a cell. In other instances, the RNAi activity occurs in a reconstituted in vitro system.

In some instances, a polynucleic acid molecule is a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In some instances, the polynucleic acid molecule is assembled from two separate polynucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (e.g., each strand comprises a nucleotide sequence that is complementary to the nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double-stranded structure, for example wherein the double-stranded region is about 19, 20, 21, 22, 23, or more base pairs); the antisense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, the polynucleic acid molecule is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the polynucleic acid molecule are linked by means of a nucleic acid based or non-nucleic acid-based linker(s).

In some cases, a polynucleic acid molecule is a polynucleotide with a duplex, asymmetric duplex, hairpin, or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In other cases, the polynucleic acid molecule is a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide is processed either in vivo or in vitro to generate an active polynucleic acid molecule capable of mediating RNAi. In additional cases, the polynucleic acid molecule also comprises a single-stranded polynucleotide having a nucleotide sequence complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such polynucleic acid molecule does not require the presence within the polynucleic acid molecule of a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide further comprises a terminal phosphate group, such as a 5′-phosphate (see for example Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate.

In some instances, an asymmetric duplex is a linear polynucleic acid molecule comprising an antisense region, a loop portion that comprises nucleotides or non-nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complimentary nucleotides to base pair with the antisense region and form a duplex with loop. For example, an asymmetric hairpin polynucleic acid molecule comprises an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g. about 19 to about 22 nucleotides) and a loop region comprising about 4 to about 8 nucleotides, and a sense region having about 3 to about 18 nucleotides that are complementary to the antisense region. In some cases, the asymmetric hairpin polynucleic acid molecule also comprises a 5′-terminal phosphate group that is chemically modified. In additional cases, the loop portion of the asymmetric hairpin polynucleic acid molecule comprises nucleotides, non-nucleotides, linker molecules, or conjugate molecules.

In some embodiments, an asymmetric duplex is a polynucleic acid molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complimentary nucleotides to base pair with the antisense region and form a duplex. For example, an asymmetric duplex polynucleic acid molecule comprises an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g. about 19 to about 22 nucleotides) and a sense region having about 3 to about 18 nucleotides that are complementary to the antisense region.

In some cases, a universal base refers to nucleotide base analogs that form base pain with each of the natural DNA/RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

Polynucleic Acid Molecule Synthesis

In some embodiments, a polynucleic acid molecule described herein is constructed using chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. For example, a polynucleic acid molecule is chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the polynucleic acid molecule and target nucleic acids. Exemplary methods include those described in: U.S. Pat. Nos. 5,142,047; 5,185,444; 5,889,136; 6,008,400; and 6,111,086; PCT Publication No. WO2009099942; or European Publication No. 1579015. Additional exemplary methods include those described in: Griffey et al., “2′-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides,” J. Med. Chem. 39(26):5100-5109 (1997)); Obika, et al. “Synthesis of 2′-O,4′-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3, -endo sugar puckering”. Tetrahedron Letters 38 (50): 8735 (1997); Koizumi, M. “ENA oligonucleotides as therapeutics”. Current opinion in molecular therapeutics 8 (2): 144-149 (2006); and Abramova et al., “Novel oligonucleotide analogues based on morpholino nucleoside subunits-antisense technologies: new chemical possibilities,” Indian Journal of Chemistry 48B:1721-1726 (2009). Alternatively, the polynucleic acid molecule is produced biologically using an expression vector into which a polynucleic acid molecule has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted polynucleic acid molecule will be of an antisense orientation to a target polynucleic acid molecule of interest).

In some embodiments, a polynucleic acid molecule is synthesized via a tandem synthesis methodology, wherein both strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate fragments or strands that hybridize and permit purification of the duplex.

In some instances, a polynucleic acid molecule is also assembled from two distinct nucleic acid strands or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the molecule.

Additional modification methods for incorporating, for example, sugar, base, and phosphate modifications include: Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010. Such publications describe general methods and strategies to determine the location of incorporation of sugar, base, and/or phosphate modifications and the like into nucleic acid molecules without modulating catalysis.

In some instances, while chemical modification of the polynucleic acid molecule internucleotide linkages with phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonate linkages improves stability, excessive modifications sometimes cause toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages in some cases is minimized. In such cases, the reduction in the concentration of these linkages lowers toxicity, and increases efficacy and specificity of these molecules.

Diseases

In some embodiments, a polynucleic acid molecule or a pharmaceutical composition described herein is used for the treatment of a disease or disorder. In some instances, the disease or disorder is a cancer. In some embodiments, a polynucleic acid molecule or a pharmaceutical composition described herein is used for the treatment of cancer. In some instances, the cancer is a solid tumor. In some instances, the cancer is a hematologic malignancy. In some instances, the cancer is a relapsed or refractory cancer, or a metastatic cancer. In some instances, the solid tumor is a relapsed or refractory solid tumor, or a metastatic solid tumor. In some cases, the hematologic malignancy is a relapsed or refractory hematologic malignancy, or a metastatic hematologic malignancy.

In some embodiments, the cancer is a solid tumor. Exemplary solid tumor includes, but is not limited to, anal cancer, appendix cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain tumor, breast cancer, cervical cancer, colon cancer, cancer of Unknown Primary (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastroenterological cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.

In some instances, a polynucleic acid molecule or a pharmaceutical composition described herein is used for the treatment of a solid tumor. In some instances, a polynucleic acid molecule or a pharmaceutical composition described herein is used for the treatment of anal cancer, appendix cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain tumor, breast cancer, cervical cancer, colon cancer, cancer of Unknown Primary (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastroenterological cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer. In some instances, the solid tumor is a relapsed or refractory solid tumor, or a metastatic solid tumor.

In some instances, the cancer is a hematologic malignancy. In some instances, the hematologic malignancy is a leukemia, a lymphoma, a myeloma, a non-Hodgkin's lymphoma, or a Hodgkin's lymphoma. In some instances, the hematologic malignancy comprises chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high risk CLL, a non-CLL/SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

In some instances, a polynucleic acid molecule or a pharmaceutical composition described herein is used for the treatment of a hematologic malignancy. In some instances, a polynucleic acid molecule or a pharmaceutical composition described herein is used for the treatment of a leukemia, a lymphoma, a myeloma, a non-Hodgkin's lymphoma, or a Hodgkin's lymphoma. In some instances, the hematologic malignancy comprises chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high risk CLL, a non-CLL/SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. In some cases, the hematologic malignancy is a relapsed or refractory hematologic malignancy, or a metastatic hematologic malignancy.

In some instances, the cancer is an EGFR-associated cancer. In some instances, a polynucleic acid molecule or a pharmaceutical composition described herein is used for the treatment of an EGFR-associated cancer. In some instances, the cancer is a solid tumor. In some instances, the cancer is a hematologic malignancy. In some instances, the solid tumor is a relapsed or refractory solid tumor, or a metastatic solid tumor. In some cases, the hematologic malignancy is a relapsed or refractory hematologic malignancy, or a metastatic hematologic malignancy. In some instances, the cancer comprises bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, glioblastoma multiforme, head and neck cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, acute myeloid leukemia, CLL, DLBCL, or multiple myeloma.

Pharmaceutical Formulation

In some embodiments, the pharmaceutical formulations described herein are administered to a subject by multiple administration routes including, but not limited to, parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal administration routes. In some instances, the pharmaceutical composition describe herein is formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular) administration. In other instances, the pharmaceutical composition describe herein is formulated for oral administration. In still other instances, the pharmaceutical composition describe herein is formulated for intranasal administration.

In some embodiments, the pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and mixed immediate- and controlled-release formulations.

In some instances, the pharmaceutical formulation includes multiparticulate formulations. In some instances, the pharmaceutical formulation includes nanoparticle formulations. In some instances, nanoparticles comprise cMAP, cyclodextrin, or lipids. In some cases, nanoparticles comprise solid lipid nanoparticles, polymeric nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions, or micellar solutions. Additional exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as with covalently attached metal chelates), nanofibers, nanohorns, nano-onions, nanorods, nanoropes, and quantum dots. In some instances, a nanoparticle is a metal nanoparticle, e.g., a nanoparticle of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium, lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium, potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, and combinations, alloys, or oxides thereof.

In some instances, a nanoparticle includes a core or a core and a shell, as in a core-shell nanoparticle.

In some instances, a nanoparticle is further coated with molecules for attachment of functional elements (e.g., with one or more of a polynucleic acid molecule or binding moiety described herein). In some instances, a coating comprises chondroitin sulfate, dextran sulfate, carboxymethyl dextran, alginic acid, pectin, carragheenan, fucoidan, agaropectin, porphyran, karaya gum, gellan gum, xanthan gum, hyaluronic acids, glucosamine, galactosamine, chitin (or chitosan), polyglutamic acid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen, α-chymotrypsin, polylysine, polyarginine, histone, protamine, ovalbumin, dextrin, or cyclodextrin. In some instances, a nanoparticle comprises a graphene-coated nanoparticle.

In some cases, a nanoparticle has at least one dimension of less than about 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm.

In some instances, the nanoparticle formulation comprises paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as with covalently attached metal chelates), nanofibers, nanohorns, nano-onions, nanorods, nanoropes or quantum dots. In some instances, a polynucleic acid molecule or a binding moiety described herein is conjugated either directly or indirectly to the nanoparticle. In some instances, at least 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more polynucleic acid molecules or binding moieties described herein are conjugated either directly or indirectly to a nanoparticle.

In some embodiments, the pharmaceutical formulation comprise a delivery vector, e.g., a recombinant vector, for the delivery of the polynucleic acid molecule into cells. In some instances, the recombinant vector is DNA plasmid. In other instances, the recombinant vector is a viral vector. Exemplary viral vectors include vectors derived from adeno-associated virus, retrovirus, adenovirus, or alphavirus. In some instances, the recombinant vectors capable of expressing the polynucleic acid molecules provide stable expression in target cells. In additional instances, viral vectors are used that provide for transient expression of polynucleic acid molecules.

In some embodiments, the pharmaceutical formulations include a carrier or carrier materials selected on the basis of compatibility with the composition disclosed herein, and the release profile properties of the desired dosage form. Exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like. See, e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

In some instances, the pharmaceutical formulations further include pH-adjusting agents or buffering agents which include acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

In some instances, the pharmaceutical formulation includes one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

In some instances, the pharmaceutical formulations further include diluent which are used to stabilize compounds because they provide a more stable environment. Salts dissolved in buffered solutions (which also provide pH control or maintenance) are utilized as diluents in the art, including, but not limited to a phosphate-buffered saline solution. In certain instances, diluents increase bulk of the composition to facilitate compression or create sufficient bulk for homogenous blend for capsule filling. Such compounds include e.g., lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®; dibasic calcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugar, such as Di-Pac® (Amstar); mannitol, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents, confectioner's sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

In some cases, the pharmaceutical formulations include disintegration agents or disintegrants to facilitate the breakup or disintegration of a substance. The term “disintegrate” includes both the dissolution and dispersion of the dosage form when contacted with gastrointestinal fluid. Examples of disintegration agents include a starch, e.g., a natural starch such as corn starch or potato starch, a pregelatinized starch such as National 1551 or Amijel®, or sodium starch glycolate such as Promogel® or Explotab®; a cellulose such as a wood product, methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, and Solka-Floc®, methylcellulose, croscarmellose, or a cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linked croscarmellose; a cross-linked starch such as sodium starch glycolate, a cross-linked polymer such as crospovidone; a cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginic acid such as sodium alginate, a clay such as Veegum® HV (magnesium aluminum silicate); a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth; sodium starch glycolate; bentonite; a natural sponge; a surfactant; a resin such as a cation-exchange resin; citrus pulp; sodium lauryl sulfate; sodium lauryl sulfate in combination starch; and the like.

In some instances, the pharmaceutical formulations include filling agents such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

Lubricants and glidants are also optionally included in the pharmaceutical formulations described herein for preventing, reducing, or inhibiting adhesion or friction of materials. Exemplary lubricants include, e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetable oil such as hydrogenated soybean oil (Sterotex®), higher fatty acids and their alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a methoxypolyethylene glycol such as Carbowax™ sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil®, a starch such as corn starch, silicone oil, a surfactant, and the like.

Plasticizers include compounds used to soften the microencapsulation material or film coatings to make them less brittle. Suitable plasticizers include, e.g., polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethyl cellulose and triacetin. Plasticizers also function as dispersing agents or wetting agents.

Solubilizers include compounds such as triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, dimethyl isosorbide, and the like.

Stabilizers include compounds such as any antioxidation agents, buffers, acids, preservatives, and the like.

Suspending agents include compounds such as polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30), vinyl pyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol (e.g., the polyethylene glycol has a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400), sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums (such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum), sugars, cellulosics (such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose), polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, and the like.

Surfactants include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic® (BASF), and the like. Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40. Sometimes, surfactants are included to enhance physical stability or for other purposes.

Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetate stearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans, and combinations thereof.

Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts, and the like.

Therapeutic Regimens

In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic applications. In some embodiments, the pharmaceutical composition is administered once per day, twice per day, three times per day, or more. The pharmaceutical composition is administered daily, every day, every alternate day, five days a week, once a week, every other week, two weeks per month, three weeks per month, once a month, twice a month, three times per month, or more. The pharmaceutical composition is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.

In some embodiments, one or more pharmaceutical compositions are administered simultaneously, sequentially, or at an interval period of time. In some embodiments, one or more pharmaceutical compositions are administered simultaneously. In some cases, one or more pharmaceutical compositions are administered sequentially. In additional cases, one or more pharmaceutical compositions are administered at an interval period of time (e.g., the first administration of a first pharmaceutical composition is on day one followed by an interval of at least 1, 2, 3, 4, 5, or more days prior to the administration of at least a second pharmaceutical composition).

In some embodiments, two or more different pharmaceutical compositions are coadministered. In some instances, the two or more different pharmaceutical compositions are coadministered simultaneously. In some cases, the two or more different pharmaceutical compositions are coadministered sequentially without a gap of time between administrations. In other cases, the two or more different pharmaceutical compositions are coadministered sequentially with a gap of about 0.5 hour, 1 hour, 2 hour, 3 hour, 12 hours, 1 day, 2 days, or more between administrations.

In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the composition is given continuously; alternatively, the dose of the composition being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”). In some instances, the length of the drug holiday varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday is from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, are optionally reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained.

In some embodiments, the amount of a given agent that correspond to such an amount varies depending upon factors such as the particular compound, the severity of the disease, the identity (e.g., weight) of the subject or host in need of treatment, but nevertheless is routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, and the subject or host being treated. In some instances, the desired dose is conveniently presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

The foregoing ranges are merely suggestive, as the number of variables in regard to an individual treatment regime is large, and considerable excursions from these recommended values are not uncommon. Such dosages are altered depending on a number of variables, not limited to the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

In some embodiments, toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it is expressed as the ratio between LD50 and ED50. Compounds exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage varies within this range depending upon the dosage form employed and the route of administration utilized.

Kits/Article of Manufacture

Disclosed herein, in certain embodiments, are kits and articles of manufacture for use with one or more of the compositions and methods described herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.

For example, the container(s) include EGFR nucleic acid molecule described herein. Such kits optionally include an identifying description or label or instructions relating to its use in the methods described herein.

A kit typically includes labels listing contents and/or instructions for use and package inserts with instructions for use. A set of instructions will also typically be included.

In one embodiment, a label is on or associated with the container. In one embodiment, a label is on a container when letters, numbers, or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein. The pack, for example, contains metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is also accompanied with a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, is the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. In one embodiment, compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

Certain Terminology

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the general description and the detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 μL” means “about 5 μL” and also “5 μL.” Generally, the term “about” includes an amount that is expected to be within experimental error, e.g., ±5%, ±10%, or ±15%.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

As used herein, the terms “individual(s)”, “subject(s)” and “patient(s)” mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly or a hospice worker).

EXAMPLES

These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

Example 1. Sequences

Table 1 illustrates target sequences described herein. Tables 2, 3, and 6 illustrate polynucleic acid molecule sequences described herein.

TABLE 1 EGFR Target Sequences 19mer pos. SEQ  in sequence of total 23mer ID hs Id # NM_005228.3 target site in NM_005228.3 NO: 68 68-86 GGCGGCCGGAGUCCCGAGCUAGC 1 71 71-89 GGCCGGAGUCCCGAGCUAGCCCC 2 72 72-90 GCCGGAGUCCCGAGCUAGCCCCG 3 73 73-91 CCGGAGUCCCGAGCUAGCCCCGG 4 74 74-92 CGGAGUCCCGAGCUAGCCCCGGC 5 75 75-93 GGAGUCCCGAGCUAGCCCCGGCG 6 76 76-94 GAGUCCCGAGCUAGCCCCGGCGG 7 78 78-96 GUCCCGAGCUAGCCCCGGCGGCC 8 114 114-132 CCGGACGACAGGCCACCUCGUCG 9 115 115-133 CGGACGACAGGCCACCUCGUCGG 10 116 116-134 GGACGACAGGCCACCUCGUCGGC 11 117 117-135 GACGACAGGCCACCUCGUCGGCG 12 118 118-136 ACGACAGGCCACCUCGUCGGCGU 13 120 120-138 GACAGGCCACCUCGUCGGCGUCC 14 121 121-139 ACAGGCCACCUCGUCGGCGUCCG 15 122 122-140 CAGGCCACCUCGUCGGCGUCCGC 16 123 123-141 AGGCCACCUCGUCGGCGUCCGCC 17 124 124-142 GGCCACCUCGUCGGCGUCCGCCC 18 125 125-143 GCCACCUCGUCGGCGUCCGCCCG 19 126 126-144 CCACCUCGUCGGCGUCCGCCCGA 20 127 127-145 CACCUCGUCGGCGUCCGCCCGAG 21 128 128-146 ACCUCGUCGGCGUCCGCCCGAGU 22 129 129-147 CCUCGUCGGCGUCCGCCCGAGUC 23 130 130-148 CUCGUCGGCGUCCGCCCGAGUCC 24 131 131-149 UCGUCGGCGUCCGCCCGAGUCCC 25 132 132-150 CGUCGGCGUCCGCCCGAGUCCCC 26 135 135-153 CGGCGUCCGCCCGAGUCCCCGCC 27 136 136-154 GGCGUCCGCCCGAGUCCCCGCCU 28 141 141-159 CCGCCCGAGUCCCCGCCUCGCCG 29 164 164-182 CCAACGCCACAACCACCGCGCAC 30 165 165-183 CAACGCCACAACCACCGCGCACG 31 166 166-184 AACGCCACAACCACCGCGCACGG 32 168 168-186 CGCCACAACCACCGCGCACGGCC 33 169 169-187 GCCACAACCACCGCGCACGGCCC 34 170 170-188 CCACAACCACCGCGCACGGCCCC 35 247 247-265 CGAUGCGACCCUCCGGGACGGCC 36 248 248-266 GAUGCGACCCUCCGGGACGGCCG 37 249 249-267 AUGCGACCCUCCGGGACGGCCGG 38 251 251-269 GCGACCCUCCGGGACGGCCGGGG 39 252 252-270 CGACCCUCCGGGACGGCCGGGGC 40 254 254-272 ACCCUCCGGGACGGCCGGGGCAG 41 329 329-347 AAAGAAAGUUUGCCAAGGCACGA 42 330 330-348 AAGAAAGUUUGCCAAGGCACGAG 43 332 332-350 GAAAGUUUGCCAAGGCACGAGUA 44 333 333-351 AAAGUUUGCCAAGGCACGAGUAA 45 334 334-352 AAGUUUGCCAAGGCACGAGUAAC 46 335 335-353 AGUUUGCCAAGGCACGAGUAACA 47 336 336-354 GUUUGCCAAGGCACGAGUAACAA 48 337 337-355 UUUGCCAAGGCACGAGUAACAAG 49 338 338-356 UUGCCAAGGCACGAGUAACAAGC 50 361 361-379 UCACGCAGUUGGGCACUUUUGAA 51 362 362-380 CACGCAGUUGGGCACUUUUGAAG 52 363 363-381 ACGCAGUUGGGCACUUUUGAAGA 53 364 364-382 CGCAGUUGGGCACUUUUGAAGAU 54 365 365-383 GCAGUUGGGCACUUUUGAAGAUC 55 366 366-384 CAGUUGGGCACUUUUGAAGAUCA 56 367 367-385 AGUUGGGCACUUUUGAAGAUCAU 57 368 368-386 GUUGGGCACUUUUGAAGAUCAUU 58 369 369-387 UUGGGCACUUUUGAAGAUCAUUU 59 377 377-395 UUUUGAAGAUCAUUUUCUCAGCC 60 379 379-397 UUGAAGAUCAUUUUCUCAGCCUC 61 380 380-398 UGAAGAUCAUUUUCUCAGCCUCC 62 385 385-403 AUCAUUUUCUCAGCCUCCAGAGG 63 394 394-412 UCAGCCUCCAGAGGAUGUUCAAU 64 396 396-414 AGCCUCCAGAGGAUGUUCAAUAA 65 397 397-415 GCCUCCAGAGGAUGUUCAAUAAC 66 401 401-419 CCAGAGGAUGUUCAAUAACUGUG 67 403 403-421 AGAGGAUGUUCAAUAACUGUGAG 68 407 407-425 GAUGUUCAAUAACUGUGAGGUGG 69 409 409-427 UGUUCAAUAACUGUGAGGUGGUC 70 410 410-428 GUUCAAUAACUGUGAGGUGGUCC 71 411 411-429 UUCAAUAACUGUGAGGUGGUCCU 72 412 412-430 UCAAUAACUGUGAGGUGGUCCUU 73 413 413-431 CAAUAACUGUGAGGUGGUCCUUG 74 414 414-432 AAUAACUGUGAGGUGGUCCUUGG 75 416 416-434 UAACUGUGAGGUGGUCCUUGGGA 76 418 418-436 ACUGUGAGGUGGUCCUUGGGAAU 77 419 419-437 CUGUGAGGUGGUCCUUGGGAAUU 78 425 425-443 GGUGGUCCUUGGGAAUUUGGAAA 79 431 431-449 CCUUGGGAAUUUGGAAAUUACCU 80 432 432-450 CUUGGGAAUUUGGAAAUUACCUA 81 433 433-451 UUGGGAAUUUGGAAAUUACCUAU 82 434 434-452 UGGGAAUUUGGAAAUUACCUAUG 83 458 458-476 GCAGAGGAAUUAUGAUCUUUCCU 84 459 459-477 CAGAGGAAUUAUGAUCUUUCCUU 85 463 463-481 GGAAUUAUGAUCUUUCCUUCUUA 86 464 464-482 GAAUUAUGAUCUUUCCUUCUUAA 87 466 466-484 AUUAUGAUCUUUCCUUCUUAAAG 88 468 468-486 UAUGAUCUUUCCUUCUUAAAGAC 89 471 471-489 GAUCUUUCCUUCUUAAAGACCAU 90 476 476-494 UUCCUUCUUAAAGACCAUCCAGG 91 477 477-495 UCCUUCUUAAAGACCAUCCAGGA 92 479 479-497 CUUCUUAAAGACCAUCCAGGAGG 93 481 481-499 UCUUAAAGACCAUCCAGGAGGUG 94 482 482-500 CUUAAAGACCAUCCAGGAGGUGG 95 492 492-510 AUCCAGGAGGUGGCUGGUUAUGU 96 493 493-511 UCCAGGAGGUGGCUGGUUAUGUC 97 494 494-512 CCAGGAGGUGGCUGGUUAUGUCC 98 495 495-513 CAGGAGGUGGCUGGUUAUGUCCU 99 496 496-514 AGGAGGUGGCUGGUUAUGUCCUC 100 497 497-515 GGAGGUGGCUGGUUAUGUCCUCA 101 499 499-517 AGGUGGCUGGUUAUGUCCUCAUU 102 520 520-538 UUGCCCUCAACACAGUGGAGCGA 103 542 542-560 AAUUCCUUUGGAAAACCUGCAGA 104 543 543-561 AUUCCUUUGGAAAACCUGCAGAU 105 550 550-568 UGGAAAACCUGCAGAUCAUCAGA 106 551 551-569 GGAAAACCUGCAGAUCAUCAGAG 107 553 553-571 AAAACCUGCAGAUCAUCAGAGGA 108 556 556-574 ACCUGCAGAUCAUCAGAGGAAAU 109 586 586-604 ACGAAAAUUCCUAUGCCUUAGCA 110 587 587-605 CGAAAAUUCCUAUGCCUUAGCAG 111 589 589-607 AAAAUUCCUAUGCCUUAGCAGUC 112 592 592-610 AUUCCUAUGCCUUAGCAGUCUUA 113 593 593-611 UUCCUAUGCCUUAGCAGUCUUAU 114 594 594-612 UCCUAUGCCUUAGCAGUCUUAUC 115 596 596-614 CUAUGCCUUAGCAGUCUUAUCUA 116 597 597-615 UAUGCCUUAGCAGUCUUAUCUAA 117 598 598-616 AUGCCUUAGCAGUCUUAUCUAAC 118 599 599-617 UGCCUUAGCAGUCUUAUCUAACU 119 600 600-618 GCCUUAGCAGUCUUAUCUAACUA 120 601 601-619 CCUUAGCAGUCUUAUCUAACUAU 121 602 602-620 CUUAGCAGUCUUAUCUAACUAUG 122 603 603-621 UUAGCAGUCUUAUCUAACUAUGA 123 604 604-622 UAGCAGUCUUAUCUAACUAUGAU 124 605 605-623 AGCAGUCUUAUCUAACUAUGAUG 125 608 608-626 AGUCUUAUCUAACUAUGAUGCAA 126 609 609-627 GUCUUAUCUAACUAUGAUGCAAA 127 610 610-628 UCUUAUCUAACUAUGAUGCAAAU 128 611 611-629 CUUAUCUAACUAUGAUGCAAAUA 129 612 612-630 UUAUCUAACUAUGAUGCAAAUAA 130 613 613-631 UAUCUAACUAUGAUGCAAAUAAA 131 614 614-632 AUCUAACUAUGAUGCAAAUAAAA 132 616 616-634 CUAACUAUGAUGCAAAUAAAACC 133 622 622-640 AUGAUGCAAAUAAAACCGGACUG 134 623 623-641 UGAUGCAAAUAAAACCGGACUGA 135 624 624-642 GAUGCAAAUAAAACCGGACUGAA 136 626 626-644 UGCAAAUAAAACCGGACUGAAGG 137 627 627-645 GCAAAUAAAACCGGACUGAAGGA 138 628 628-646 CAAAUAAAACCGGACUGAAGGAG 139 630 630-648 AAUAAAACCGGACUGAAGGAGCU 140 631 631-649 AUAAAACCGGACUGAAGGAGCUG 141 632 632-650 UAAAACCGGACUGAAGGAGCUGC 142 633 633-651 AAAACCGGACUGAAGGAGCUGCC 143 644 644-662 GAAGGAGCUGCCCAUGAGAAAUU 144 665 665-683 UUUACAGGAAAUCCUGCAUGGCG 145 668 668-686 ACAGGAAAUCCUGCAUGGCGCCG 146 669 669-687 CAGGAAAUCCUGCAUGGCGCCGU 147 670 670-688 AGGAAAUCCUGCAUGGCGCCGUG 148 671 671-689 GGAAAUCCUGCAUGGCGCCGUGC 149 672 672-690 GAAAUCCUGCAUGGCGCCGUGCG 150 674 674-692 AAUCCUGCAUGGCGCCGUGCGGU 151 676 676-694 UCCUGCAUGGCGCCGUGCGGUUC 152 677 677-695 CCUGCAUGGCGCCGUGCGGUUCA 153 678 678-696 CUGCAUGGCGCCGUGCGGUUCAG 154 680 680-698 GCAUGGCGCCGUGCGGUUCAGCA 155 681 681-699 CAUGGCGCCGUGCGGUUCAGCAA 156 682 682-700 AUGGCGCCGUGCGGUUCAGCAAC 157 683 683-701 UGGCGCCGUGCGGUUCAGCAACA 158 684 684-702 GGCGCCGUGCGGUUCAGCAACAA 159 685 685-703 GCGCCGUGCGGUUCAGCAACAAC 160 686 686-704 CGCCGUGCGGUUCAGCAACAACC 161 688 688-706 CCGUGCGGUUCAGCAACAACCCU 162 690 690-708 GUGCGGUUCAGCAACAACCCUGC 163 692 692-710 GCGGUUCAGCAACAACCCUGCCC 164 698 698-716 CAGCAACAACCCUGCCCUGUGCA 165 700 700-718 GCAACAACCCUGCCCUGUGCAAC 166 719 719-737 CAACGUGGAGAGCAUCCAGUGGC 167 720 720-738 AACGUGGAGAGCAUCCAGUGGCG 168 721 721-739 ACGUGGAGAGCAUCCAGUGGCGG 169 724 724-742 UGGAGAGCAUCCAGUGGCGGGAC 170 725 725-743 GGAGAGCAUCCAGUGGCGGGACA 171 726 726-744 GAGAGCAUCCAGUGGCGGGACAU 172 733 733-751 UCCAGUGGCGGGACAUAGUCAGC 173 734 734-752 CCAGUGGCGGGACAUAGUCAGCA 174 736 736-754 AGUGGCGGGACAUAGUCAGCAGU 175 737 737-755 GUGGCGGGACAUAGUCAGCAGUG 176 763 763-781 UUCUCAGCAACAUGUCGAUGGAC 177 765 765-783 CUCAGCAACAUGUCGAUGGACUU 178 766 766-784 UCAGCAACAUGUCGAUGGACUUC 179 767 767-785 CAGCAACAUGUCGAUGGACUUCC 180 769 769-787 GCAACAUGUCGAUGGACUUCCAG 181 770 770-788 CAACAUGUCGAUGGACUUCCAGA 182 771 771-789 AACAUGUCGAUGGACUUCCAGAA 183 772 772-790 ACAUGUCGAUGGACUUCCAGAAC 184 775 775-793 UGUCGAUGGACUUCCAGAACCAC 185 789 789-807 CAGAACCACCUGGGCAGCUGCCA 186 798 798-816 CUGGGCAGCUGCCAAAAGUGUGA 187 800 800-818 GGGCAGCUGCCAAAAGUGUGAUC 188 805 805-823 GCUGCCAAAAGUGUGAUCCAAGC 189 806 806-824 CUGCCAAAAGUGUGAUCCAAGCU 190 807 807-825 UGCCAAAAGUGUGAUCCAAGCUG 191 810 810-828 CAAAAGUGUGAUCCAAGCUGUCC 192 814 814-832 AGUGUGAUCCAAGCUGUCCCAAU 193 815 815-833 GUGUGAUCCAAGCUGUCCCAAUG 194 817 817-835 GUGAUCCAAGCUGUCCCAAUGGG 195 818 818-836 UGAUCCAAGCUGUCCCAAUGGGA 196 819 819-837 GAUCCAAGCUGUCCCAAUGGGAG 197 820 820-838 AUCCAAGCUGUCCCAAUGGGAGC 198 821 821-839 UCCAAGCUGUCCCAAUGGGAGCU 199 823 823-841 CAAGCUGUCCCAAUGGGAGCUGC 200 826 826-844 GCUGUCCCAAUGGGAGCUGCUGG 201 847 847-865 GGGGUGCAGGAGAGGAGAACUGC 202 871 871-889 AGAAACUGACCAAAAUCAUCUGU 203 872 872-890 GAAACUGACCAAAAUCAUCUGUG 204 873 873-891 AAACUGACCAAAAUCAUCUGUGC 205 877 877-895 UGACCAAAAUCAUCUGUGCCCAG 206 878 878-896 GACCAAAAUCAUCUGUGCCCAGC 207 881 881-899 CAAAAUCAUCUGUGCCCAGCAGU 208 890 890-908 CUGUGCCCAGCAGUGCUCCGGGC 209 892 892-910 GUGCCCAGCAGUGCUCCGGGCGC 210 929 929-947 CCCCAGUGACUGCUGCCACAACC 211 930 930-948 CCCAGUGACUGCUGCCACAACCA 212 979 979-997 GGGAGAGCGACUGCCUGGUCUGC 213 980 980-998 GGAGAGCGACUGCCUGGUCUGCC 214 981 981-999 GAGAGCGACUGCCUGGUCUGCCG 215 982  982-1000 AGAGCGACUGCCUGGUCUGCCGC 216 983  983-1001 GAGCGACUGCCUGGUCUGCCGCA 217 984  984-1002 AGCGACUGCCUGGUCUGCCGCAA 218 989  989-1007 CUGCCUGGUCUGCCGCAAAUUCC 219 990  990-1008 UGCCUGGUCUGCCGCAAAUUCCG 220 991  991-1009 GCCUGGUCUGCCGCAAAUUCCGA 221 992  992-1010 CCUGGUCUGCCGCAAAUUCCGAG 222 994  994-1012 UGGUCUGCCGCAAAUUCCGAGAC 223 995  995-1013 GGUCUGCCGCAAAUUCCGAGACG 224 996  996-1014 GUCUGCCGCAAAUUCCGAGACGA 225 997  997-1015 UCUGCCGCAAAUUCCGAGACGAA 226 999  999-1017 UGCCGCAAAUUCCGAGACGAAGC 227 1004 1004-1022 CAAAUUCCGAGACGAAGCCACGU 228 1005 1005-1023 AAAUUCCGAGACGAAGCCACGUG 229 1006 1006-1024 AAUUCCGAGACGAAGCCACGUGC 230 1007 1007-1025 AUUCCGAGACGAAGCCACGUGCA 231 1008 1008-1026 UUCCGAGACGAAGCCACGUGCAA 232 1010 1010-1028 CCGAGACGAAGCCACGUGCAAGG 233 1013 1013-1031 AGACGAAGCCACGUGCAAGGACA 234 1014 1014-1032 GACGAAGCCACGUGCAAGGACAC 235 1015 1015-1033 ACGAAGCCACGUGCAAGGACACC 236 1016 1016-1034 CGAAGCCACGUGCAAGGACACCU 237 1040 1040-1058 CCCCCCACUCAUGCUCUACAACC 238 1042 1042-1060 CCCCACUCAUGCUCUACAACCCC 239 1044 1044-1062 CCACUCAUGCUCUACAACCCCAC 240 1047 1047-1065 CUCAUGCUCUACAACCCCACCAC 241 1071 1071-1089 UACCAGAUGGAUGUGAACCCCGA 242 1073 1073-1091 CCAGAUGGAUGUGAACCCCGAGG 243 1074 1074-1092 CAGAUGGAUGUGAACCCCGAGGG 244 1075 1075-1093 AGAUGGAUGUGAACCCCGAGGGC 245 1077 1077-1095 AUGGAUGUGAACCCCGAGGGCAA 246 1078 1078-1096 UGGAUGUGAACCCCGAGGGCAAA 247 1080 1080-1098 GAUGUGAACCCCGAGGGCAAAUA 248 1084 1084-1102 UGAACCCCGAGGGCAAAUACAGC 249 1085 1085-1103 GAACCCCGAGGGCAAAUACAGCU 250 1087 1087-1105 ACCCCGAGGGCAAAUACAGCUUU 251 1088 1088-1106 CCCCGAGGGCAAAUACAGCUUUG 252 1089 1089-1107 CCCGAGGGCAAAUACAGCUUUGG 253 1096 1096-1114 GCAAAUACAGCUUUGGUGCCACC 254 1097 1097-1115 CAAAUACAGCUUUGGUGCCACCU 255 1098 1098-1116 AAAUACAGCUUUGGUGCCACCUG 256 1104 1104-1122 AGCUUUGGUGCCACCUGCGUGAA 257 1106 1106-1124 CUUUGGUGCCACCUGCGUGAAGA 258 1112 1112-1130 UGCCACCUGCGUGAAGAAGUGUC 259 1116 1116-1134 ACCUGCGUGAAGAAGUGUCCCCG 260 1117 1117-1135 CCUGCGUGAAGAAGUGUCCCCGU 261 1118 1118-1136 CUGCGUGAAGAAGUGUCCCCGUA 262 1119 1119-1137 UGCGUGAAGAAGUGUCCCCGUAA 263 1120 1120-1138 GCGUGAAGAAGUGUCCCCGUAAU 264 1121 1121-1139 CGUGAAGAAGUGUCCCCGUAAUU 265 1122 1122-1140 GUGAAGAAGUGUCCCCGUAAUUA 266 1123 1123-1141 UGAAGAAGUGUCCCCGUAAUUAU 267 1124 1124-1142 GAAGAAGUGUCCCCGUAAUUAUG 268 1125 1125-1143 AAGAAGUGUCCCCGUAAUUAUGU 269 1126 1126-1144 AGAAGUGUCCCCGUAAUUAUGUG 270 1127 1127-1145 GAAGUGUCCCCGUAAUUAUGUGG 271 1128 1128-1146 AAGUGUCCCCGUAAUUAUGUGGU 272 1129 1129-1147 AGUGUCCCCGUAAUUAUGUGGUG 273 1130 1130-1148 GUGUCCCCGUAAUUAUGUGGUGA 274 1132 1132-1150 GUCCCCGUAAUUAUGUGGUGACA 275 1134 1134-1152 CCCCGUAAUUAUGUGGUGACAGA 276 1136 1136-1154 CCGUAAUUAUGUGGUGACAGAUC 277 1137 1137-1155 CGUAAUUAUGUGGUGACAGAUCA 278 1138 1138-1156 GUAAUUAUGUGGUGACAGAUCAC 279 1139 1139-1157 UAAUUAUGUGGUGACAGAUCACG 280 1140 1140-1158 AAUUAUGUGGUGACAGAUCACGG 281 1142 1142-1160 UUAUGUGGUGACAGAUCACGGCU 282 1145 1145-1163 UGUGGUGACAGAUCACGGCUCGU 283 1147 1147-1165 UGGUGACAGAUCACGGCUCGUGC 284 1148 1148-1166 GGUGACAGAUCACGGCUCGUGCG 285 1149 1149-1167 GUGACAGAUCACGGCUCGUGCGU 286 1150 1150-1168 UGACAGAUCACGGCUCGUGCGUC 287 1151 1151-1169 GACAGAUCACGGCUCGUGCGUCC 288 1152 1152-1170 ACAGAUCACGGCUCGUGCGUCCG 289 1153 1153-1171 CAGAUCACGGCUCGUGCGUCCGA 290 1154 1154-1172 AGAUCACGGCUCGUGCGUCCGAG 291 1155 1155-1173 GAUCACGGCUCGUGCGUCCGAGC 292 1156 1156-1174 AUCACGGCUCGUGCGUCCGAGCC 293 1157 1157-1175 UCACGGCUCGUGCGUCCGAGCCU 294 1160 1160-1178 CGGCUCGUGCGUCCGAGCCUGUG 295 1200 1200-1218 AUGGAGGAAGACGGCGUCCGCAA 296 1201 1201-1219 UGGAGGAAGACGGCGUCCGCAAG 297 1203 1203-1221 GAGGAAGACGGCGUCCGCAAGUG 298 1204 1204-1222 AGGAAGACGGCGUCCGCAAGUGU 299 1205 1205-1223 GGAAGACGGCGUCCGCAAGUGUA 300 1207 1207-1225 AAGACGGCGUCCGCAAGUGUAAG 301 1208 1208-1226 AGACGGCGUCCGCAAGUGUAAGA 302 1211 1211-1229 CGGCGUCCGCAAGUGUAAGAAGU 303 1212 1212-1230 GGCGUCCGCAAGUGUAAGAAGUG 304 1213 1213-1231 GCGUCCGCAAGUGUAAGAAGUGC 305 1214 1214-1232 CGUCCGCAAGUGUAAGAAGUGCG 306 1215 1215-1233 GUCCGCAAGUGUAAGAAGUGCGA 307 1216 1216-1234 UCCGCAAGUGUAAGAAGUGCGAA 308 1217 1217-1235 CCGCAAGUGUAAGAAGUGCGAAG 309 1219 1219-1237 GCAAGUGUAAGAAGUGCGAAGGG 310 1220 1220-1238 CAAGUGUAAGAAGUGCGAAGGGC 311 1221 1221-1239 AAGUGUAAGAAGUGCGAAGGGCC 312 1222 1222-1240 AGUGUAAGAAGUGCGAAGGGCCU 313 1223 1223-1241 GUGUAAGAAGUGCGAAGGGCCUU 314 1224 1224-1242 UGUAAGAAGUGCGAAGGGCCUUG 315 1225 1225-1243 GUAAGAAGUGCGAAGGGCCUUGC 316 1226 1226-1244 UAAGAAGUGCGAAGGGCCUUGCC 317 1229 1229-1247 GAAGUGCGAAGGGCCUUGCCGCA 318 1230 1230-1248 AAGUGCGAAGGGCCUUGCCGCAA 319 1231 1231-1249 AGUGCGAAGGGCCUUGCCGCAAA 320 1232 1232-1250 GUGCGAAGGGCCUUGCCGCAAAG 321 1233 1233-1251 UGCGAAGGGCCUUGCCGCAAAGU 322 1235 1235-1253 CGAAGGGCCUUGCCGCAAAGUGU 323 1236 1236-1254 GAAGGGCCUUGCCGCAAAGUGUG 324 1237 1237-1255 AAGGGCCUUGCCGCAAAGUGUGU 325 1238 1238-1256 AGGGCCUUGCCGCAAAGUGUGUA 326 1239 1239-1257 GGGCCUUGCCGCAAAGUGUGUAA 327 1241 1241-1259 GCCUUGCCGCAAAGUGUGUAACG 328 1261 1261-1279 ACGGAAUAGGUAUUGGUGAAUUU 329 1262 1262-1280 CGGAAUAGGUAUUGGUGAAUUUA 330 1263 1263-1281 GGAAUAGGUAUUGGUGAAUUUAA 331 1264 1264-1282 GAAUAGGUAUUGGUGAAUUUAAA 332 1266 1266-1284 AUAGGUAUUGGUGAAUUUAAAGA 333 1267 1267-1285 UAGGUAUUGGUGAAUUUAAAGAC 334 1289 1289-1307 CUCACUCUCCAUAAAUGCUACGA 335 1313 1313-1331 UAUUAAACACUUCAAAAACUGCA 336 1320 1320-1338 CACUUCAAAAACUGCACCUCCAU 337 1321 1321-1339 ACUUCAAAAACUGCACCUCCAUC 338 1322 1322-1340 CUUCAAAAACUGCACCUCCAUCA 339 1323 1323-1341 UUCAAAAACUGCACCUCCAUCAG 340 1324 1324-1342 UCAAAAACUGCACCUCCAUCAGU 341 1328 1328-1346 AAACUGCACCUCCAUCAGUGGCG 342 1332 1332-1350 UGCACCUCCAUCAGUGGCGAUCU 343 1333 1333-1351 GCACCUCCAUCAGUGGCGAUCUC 344 1335 1335-1353 ACCUCCAUCAGUGGCGAUCUCCA 345 1338 1338-1356 UCCAUCAGUGGCGAUCUCCACAU 346 1344 1344-1362 AGUGGCGAUCUCCACAUCCUGCC 347 1345 1345-1363 GUGGCGAUCUCCACAUCCUGCCG 348 1346 1346-1364 UGGCGAUCUCCACAUCCUGCCGG 349 1347 1347-1365 GGCGAUCUCCACAUCCUGCCGGU 350 1348 1348-1366 GCGAUCUCCACAUCCUGCCGGUG 351 1353 1353-1371 CUCCACAUCCUGCCGGUGGCAUU 352 1354 1354-1372 UCCACAUCCUGCCGGUGGCAUUU 353 1355 1355-1373 CCACAUCCUGCCGGUGGCAUUUA 354 1357 1357-1375 ACAUCCUGCCGGUGGCAUUUAGG 355 1360 1360-1378 UCCUGCCGGUGGCAUUUAGGGGU 356 1361 1361-1379 CCUGCCGGUGGCAUUUAGGGGUG 357 1362 1362-1380 CUGCCGGUGGCAUUUAGGGGUGA 358 1363 1363-1381 UGCCGGUGGCAUUUAGGGGUGAC 359 1366 1366-1384 CGGUGGCAUUUAGGGGUGACUCC 360 1369 1369-1387 UGGCAUUUAGGGGUGACUCCUUC 361 1370 1370-1388 GGCAUUUAGGGGUGACUCCUUCA 362 1371 1371-1389 GCAUUUAGGGGUGACUCCUUCAC 363 1372 1372-1390 CAUUUAGGGGUGACUCCUUCACA 364 1373 1373-1391 AUUUAGGGGUGACUCCUUCACAC 365 1374 1374-1392 UUUAGGGGUGACUCCUUCACACA 366 1404 1404-1422 CCUCUGGAUCCACAGGAACUGGA 367 1408 1408-1426 UGGAUCCACAGGAACUGGAUAUU 368 1409 1409-1427 GGAUCCACAGGAACUGGAUAUUC 369 1411 1411-1429 AUCCACAGGAACUGGAUAUUCUG 370 1412 1412-1430 UCCACAGGAACUGGAUAUUCUGA 371 1419 1419-1437 GAACUGGAUAUUCUGAAAACCGU 372 1426 1426-1444 AUAUUCUGAAAACCGUAAAGGAA 373 1427 1427-1445 UAUUCUGAAAACCGUAAAGGAAA 374 1430 1430-1448 UCUGAAAACCGUAAAGGAAAUCA 375 1431 1431-1449 CUGAAAACCGUAAAGGAAAUCAC 376

TABLE 2 EGFR siRNA Sequences Sequence position SEQ SEQ  in sense strand sequence  ID antisense strand sequence ID hs Id # NM_005228.3 (5′-3′) NO: (5′-3′) NO: 68 68-86 CGGCCGGAGUCCCGAG 377 UAGCUCGGGACUCCGGC 378 CUATT CGTT 71 71-89 CCGGAGUCCCGAGCUA 379 GGCUAGCUCGGGACUCC 380 GCCTT GGTT 72 72-90 CGGAGUCCCGAGCUAG 381 GGGCUAGCUCGGGACUC 382 CCCTT CGTT 73 73-91 GGAGUCCCGAGCUAGC 383 GGGGCUAGCUCGGGACU 384 CCCTT CCTT 74 74-92 GAGUCCCGAGCUAGCC 385 CGGGGCUAGCUCGGGAC 386 CCGTT UCTT 75 75-93 AGUCCCGAGCUAGCCC 387 CCGGGGCUAGCUCGGGA 388 CGGTT CUTT 76 76-94 GUCCCGAGCUAGCCCC 389 GCCGGGGCUAGCUCGGG 390 GGCTT ACTT 78 78-96 CCCGAGCUAGCCCCGG 391 CCGCCGGGGCUAGCUCG 392 CGGTT GGTT 114 114-132 GGACGACAGGCCACCU 393 ACGAGGUGGCCUGUCGU 394 CGUTT CCTT 115 115-133 GACGACAGGCCACCUC 395 GACGAGGUGGCCUGUCG 396 GUCTT UCTT 116 116-134 ACGACAGGCCACCUCG 397 CGACGAGGUGGCCUGUC 398 UCGTT GUTT 117 117-135 CGACAGGCCACCUCGU 399 CCGACGAGGUGGCCUGU 400 CGGTT CGTT 118 118-136 GACAGGCCACCUCGUC 401 GCCGACGAGGUGGCCUG 402 GGCTT UCTT 120 120-138 CAGGCCACCUCGUCGG 403 ACGCCGACGAGGUGGCC 404 CGUTT UGTT 121 121-139 AGGCCACCUCGUCGGC 405 GACGCCGACGAGGUGGC 406 GUCTT CUTT 122 122-140 GGCCACCUCGUCGGCG 407 GGACGCCGACGAGGUGG 408 UCCTT CCTT 123 123-141 GCCACCUCGUCGGCGU 409 CGGACGCCGACGAGGUG 410 CCGTT GCTT 124 124-142 CCACCUCGUCGGCGUC 411 GCGGACGCCGACGAGGU 412 CGCTT GGTT 125 125-143 CACCUCGUCGGCGUCC 413 GGCGGACGCCGACGAGG 414 GCCTT UGTT 126 126-144 ACCUCGUCGGCGUCCG 415 GGGCGGACGCCGACGAG 416 CCCTT GUTT 127 127-145 CCUCGUCGGCGUCCGC 417 CGGGCGGACGCCGACGA 418 CCGTT GGTT 128 128-146 CUCGUCGGCGUCCGCC 419 UCGGGCGGACGCCGACG 420 CGATT AGTT 129 129-147 UCGUCGGCGUCCGCCC 421 CUCGGGCGGACGCCGAC 422 GAGTT GATT 130 130-148 CGUCGGCGUCCGCCCG 423 ACUCGGGCGGACGCCGA 424 AGUTT CGTT 131 131-149 GUCGGCGUCCGCCCGA 425 GACUCGGGCGGACGCCG 426 GUCTT ACTT 132 132-150 UCGGCGUCCGCCCGAG 427 GGACUCGGGCGGACGCC 428 UCCTT GATT 135 135-153 GCGUCCGCCCGAGUCC 429 CGGGGACUCGGGCGGAC 430 CCGTT GCTT 136 136-154 CGUCCGCCCGAGUCCC 431 GCGGGGACUCGGGCGGA 432 CGCTT CGTT 141 141-159 GCCCGAGUCCCCGCCU 433 GCGAGGCGGGGACUCGG 434 CGCTT GCTT 164 164-182 AACGCCACAACCACCG 435 GCGCGGUGGUUGUGGC 436 CGCTT GUUTT 165 165-183 ACGCCACAACCACCGC 437 UGCGCGGUGGUUGUGG 438 GCATT CGUTT 166 166-184 CGCCACAACCACCGCG 439 GUGCGCGGUGGUUGUG 440 CACTT GCGTT 168 168-186 CCACAACCACCGCGCA 441 CCGUGCGCGGUGGUUGU 442 CGGTT GGTT 169 169-187 CACAACCACCGCGCAC 443 GCCGUGCGCGGUGGUUG 444 GGCTT UGTT 170 170-188 ACAACCACCGCGCACG 445 GGCCGUGCGCGGUGGUU 446 GCCTT GUTT 247 247-265 AUGCGACCCUCCGGGA 447 CCGUCCCGGAGGGUCGC 448 CGGTT AUTT 248 248-266 UGCGACCCUCCGGGAC 449 GCCGUCCCGGAGGGUCG 450 GGCTT CATT 249 249-267 GCGACCCUCCGGGACG 451 GGCCGUCCCGGAGGGUC 452 GCCTT GCTT 251 251-269 GACCCUCCGGGACGGC 453 CCGGCCGUCCCGGAGGG 454 CGGTT UCTT 252 252-270 ACCCUCCGGGACGGCC 455 CCCGGCCGUCCCGGAGG 456 GGGTT GUTT 254 254-272 CCUCCGGGACGGCCGG 457 GCCCCGGCCGUCCCGGA 458 GGCTT GGTT 329 329-347 AGAAAGUUUGCCAAG 459 GUGCCUUGGCAAACUUU 460 GCACTT CUTT 330 330-348 GAAAGUUUGCCAAGG 461 CGUGCCUUGGCAAACUU 462 CACGTT UCTT 332 332-350 AAGUUUGCCAAGGCA 463 CUCGUGCCUUGGCAAAC 464 CGAGTT UUTT 333 333-351 AGUUUGCCAAGGCAC 465 ACUCGUGCCUUGGCAAA 466 GAGUTT CUTT 334 334-352 GUUUGCCAAGGCACG 467 UACUCGUGCCUUGGCAA 468 AGUATT ACTT 335 335-353 UUUGCCAAGGCACGA 469 UUACUCGUGCCUUGGCA 470 GUAATT AATT 336 336-354 UUGCCAAGGCACGAG 471 GUUACUCGUGCCUUGGC 472 UAACTT AATT 337 337-355 UGCCAAGGCACGAGU 473 UGUUACUCGUGCCUUGG 474 AACATT CATT 338 338-356 GCCAAGGCACGAGUA 475 UUGUUACUCGUGCCUUG 476 ACAATT GCTT 361 361-379 ACGCAGUUGGGCACU 477 CAAAAGUGCCCAACUGC 478 UUUGTT GUTT 362 362-380 CGCAGUUGGGCACUU 479 UCAAAAGUGCCCAACUG 480 UUGATT CGTT 363 363-381 GCAGUUGGGCACUUU 481 UUCAAAAGUGCCCAACU 482 UGAATT GCTT 364 364-382 CAGUUGGGCACUUUU 483 CUUCAAAAGUGCCCAAC 484 GAAGTT UGTT 365 365-383 AGUUGGGCACUUUUG 485 UCUUCAAAAGUGCCCAA 486 AAGATT CUTT 366 366-384 GUUGGGCACUUUUGA 487 AUCUUCAAAAGUGCCCA 488 AGAUTT ACTT 367 367-385 UUGGGCACUUUUGAA 489 GAUCUUCAAAAGUGCCC 490 GAUCTT AATT 368 368-386 UGGGCACUUUUGAAG 491 UGAUCUUCAAAAGUGCC 492 AUCATT CATT 369 369-387 GGGCACUUUUGAAGA 493 AUGAUCUUCAAAAGUG 494 UCAUTT CCCTT 377 377-395 UUGAAGAUCAUUUUC 495 CUGAGAAAAUGAUCUU 496 UCAGTT CAATT 379 379-397 GAAGAUCAUUUUCUC 497 GGCUGAGAAAAUGAUC 498 AGCCTT UUCTT 380 380-398 AAGAUCAUUUUCUCA 499 AGGCUGAGAAAAUGAU 500 GCCUTT CUUTT 385 385-403 CAUUUUCUCAGCCUCC 501 UCUGGAGGCUGAGAAA 502 AGATT AUGTT 394 394-412 AGCCUCCAGAGGAUG 503 UGAACAUCCUCUGGAGG 504 UUCATT CUTT 396 396-414 CCUCCAGAGGAUGUUC 505 AUUGAACAUCCUCUGGA 506 AAUTT GGTT 397 397-415 CUCCAGAGGAUGUUC 507 UAUUGAACAUCCUCUGG 508 AAUATT AGTT 401 401-419 AGAGGAUGUUCAAUA 509 CAGUUAUUGAACAUCCU 510 ACUGTT CUTT 403 403-421 AGGAUGUUCAAUAAC 511 CACAGUUAUUGAACAUC 512 UGUGTT CUTT 407 407-425 UGUUCAAUAACUGUG 513 ACCUCACAGUUAUUGAA 514 AGGUTT CATT 409 409-427 UUCAAUAACUGUGAG 515 CCACCUCACAGUUAUUG 516 GUGGTT AATT 410 410-428 UCAAUAACUGUGAGG 517 ACCACCUCACAGUUAUU 518 UGGUTT GATT 411 411-429 CAAUAACUGUGAGGU 519 GACCACCUCACAGUUAU 520 GGUCTT UGTT 412 412-430 AAUAACUGUGAGGUG 521 GGACCACCUCACAGUUA 522 GUCCTT UUTT 413 413-431 AUAACUGUGAGGUGG 523 AGGACCACCUCACAGUU 524 UCCUTT AUTT 414 414-432 UAACUGUGAGGUGGU 525 AAGGACCACCUCACAGU 526 CCUUTT UATT 416 416-434 ACUGUGAGGUGGUCC 527 CCAAGGACCACCUCACA 528 UUGGTT GUTT 418 418-436 UGUGAGGUGGUCCUU 529 UCCCAAGGACCACCUCA 530 GGGATT CATT 419 419-437 GUGAGGUGGUCCUUG 531 UUCCCAAGGACCACCUC 532 GGAATT ACTT 425 425-443 UGGUCCUUGGGAAUU 533 UCCAAAUUCCCAAGGAC 534 UGGATT CATT 431 431-449 UUGGGAAUUUGGAAA 535 GUAAUUUCCAAAUUCCC 536 UUACTT AATT 432 432-450 UGGGAAUUUGGAAAU 537 GGUAAUUUCCAAAUUCC 538 UACCTT CATT 433 433-451 GGGAAUUUGGAAAUU 539 AGGUAAUUUCCAAAUU 540 ACCUTT CCCTT 434 434-452 GGAAUUUGGAAAUUA 541 UAGGUAAUUUCCAAAU 542 CCUATT UCCTT 458 458-476 AGAGGAAUUAUGAUC 543 GAAAGAUCAUAAUUCC 544 UUUCTT UCUTT 459 459-477 GAGGAAUUAUGAUCU 545 GGAAAGAUCAUAAUUC 546 UUCCTT CUCTT 463 463-481 AAUUAUGAUCUUUCC 547 AGAAGGAAAGAUCAUA 548 UUCUTT AUUTT 464 464-482 AUUAUGAUCUUUCCU 549 AAGAAGGAAAGAUCAU 550 UCUUTT AAUTT 466 466-484 UAUGAUCUUUCCUUC 551 UUAAGAAGGAAAGAUC 552 UUAATT AUATT 468 468-486 UGAUCUUUCCUUCUU 553 CUUUAAGAAGGAAAGA 554 AAAGTT UCATT 471 471-489 UCUUUCCUUCUUAAA 555 GGUCUUUAAGAAGGAA 556 GACCTT AGATT 476 476-494 CCUUCUUAAAGACCAU 557 UGGAUGGUCUUUAAGA 558 CCATT AGGTT 477 477-495 CUUCUUAAAGACCAUC 559 CUGGAUGGUCUUUAAG 560 CAGTT AAGTT 479 479-497 UCUUAAAGACCAUCCA 561 UCCUGGAUGGUCUUUA 562 GGATT AGATT 481 481-499 UUAAAGACCAUCCAG 563 CCUCCUGGAUGGUCUUU 564 GAGGTT AATT 482 482-500 UAAAGACCAUCCAGG 565 ACCUCCUGGAUGGUCUU 566 AGGUTT UATT 492 492-510 CCAGGAGGUGGCUGG 567 AUAACCAGCCACCUCCU 568 UUAUTT GGTT 493 493-511 CAGGAGGUGGCUGGU 569 CAUAACCAGCCACCUCC 570 UAUGTT UGTT 494 494-512 AGGAGGUGGCUGGUU 571 ACAUAACCAGCCACCUC 572 AUGUTT CUTT 495 495-513 GGAGGUGGCUGGUUA 573 GACAUAACCAGCCACCU 574 UGUCTT CCTT 496 496-514 GAGGUGGCUGGUUAU 575 GGACAUAACCAGCCACC 576 GUCCTT UCTT 497 497-515 AGGUGGCUGGUUAUG 577 AGGACAUAACCAGCCAC 578 UCCUTT CUTT 499 499-517 GUGGCUGGUUAUGUC 579 UGAGGACAUAACCAGCC 580 CUCATT ACTT 520 520-538 GCCCUCAACACAGUGG 581 GCUCCACUGUGUUGAGG 582 AGCTT GCTT 542 542-560 UUCCUUUGGAAAACC 583 UGCAGGUUUUCCAAAG 584 UGCATT GAATT 543 543-561 UCCUUUGGAAAACCU 585 CUGCAGGUUUUCCAAAG 586 GCAGTT GATT 550 550-568 GAAAACCUGCAGAUC 587 UGAUGAUCUGCAGGUU 588 AUCATT UUCTT 551 551-569 AAAACCUGCAGAUCA 589 CUGAUGAUCUGCAGGU 590 UCAGTT UUUTT 553 553-571 AACCUGCAGAUCAUCA 591 CUCUGAUGAUCUGCAGG 592 GAGTT UUTT 556 556-574 CUGCAGAUCAUCAGA 593 UUCCUCUGAUGAUCUGC 594 GGAATT AGTT 586 586-604 GAAAAUUCCUAUGCC 595 CUAAGGCAUAGGAAUU 596 UUAGTT UUCTT 587 587-605 AAAAUUCCUAUGCCU 597 GCUAAGGCAUAGGAAU 598 UAGCTT UUUTT 589 589-607 AAUUCCUAUGCCUUA 599 CUGCUAAGGCAUAGGA 600 GCAGTT AUUTT 592 592-610 UCCUAUGCCUUAGCAG 601 AGACUGCUAAGGCAUA 602 UCUTT GGATT 593 593-611 CCUAUGCCUUAGCAGU 603 AAGACUGCUAAGGCAU 604 CUUTT AGGTT 594 594-612 CUAUGCCUUAGCAGUC 605 UAAGACUGCUAAGGCA 606 UUATT UAGTT 596 596-614 AUGCCUUAGCAGUCU 607 GAUAAGACUGCUAAGG 608 UAUCTT CAUTT 597 597-615 UGCCUUAGCAGUCUU 609 AGAUAAGACUGCUAAG 610 AUCUTT GCATT 598 598-616 GCCUUAGCAGUCUUA 611 UAGAUAAGACUGCUAA 612 UCUATT GGCTT 599 599-617 CCUUAGCAGUCUUAUC 613 UUAGAUAAGACUGCUA 614 UAATT AGGTT 600 600-618 CUUAGCAGUCUUAUC 615 GUUAGAUAAGACUGCU 616 UAACTT AAGTT 601 601-619 UUAGCAGUCUUAUCU 617 AGUUAGAUAAGACUGC 618 AACUTT UAATT 602 602-620 UAGCAGUCUUAUCUA 619 UAGUUAGAUAAGACUG 620 ACUATT CUATT 603 603-621 AGCAGUCUUAUCUAA 621 AUAGUUAGAUAAGACU 622 CUAUTT GCUTT 604 604-622 GCAGUCUUAUCUAAC 623 CAUAGUUAGAUAAGAC 624 UAUGTT UGCTT 605 605-623 CAGUCUUAUCUAACU 625 UCAUAGUUAGAUAAGA 626 AUGATT CUGTT 608 608-626 UCUUAUCUAACUAUG 627 GCAUCAUAGUUAGAUA 628 AUGCTT AGATT 609 609-627 CUUAUCUAACUAUGA 629 UGCAUCAUAGUUAGAU 630 UGCATT AAGTT 610 610-628 UUAUCUAACUAUGAU 631 UUGCAUCAUAGUUAGA 632 GCAATT UAATT 611 611-629 UAUCUAACUAUGAUG 633 UUUGCAUCAUAGUUAG 634 CAAATT AUATT 612 612-630 AUCUAACUAUGAUGC 635 AUUUGCAUCAUAGUUA 636 AAAUTT GAUTT 613 613-631 UCUAACUAUGAUGCA 637 UAUUUGCAUCAUAGUU 638 AAUATT AGATT 614 614-632 CUAACUAUGAUGCAA 639 UUAUUUGCAUCAUAGU 640 AUAATT UAGTT 616 616-634 AACUAUGAUGCAAAU 641 UUUUAUUUGCAUCAUA 642 AAAATT GUUTT 622 622-640 GAUGCAAAUAAAACC 643 GUCCGGUUUUAUUUGC 644 GGACTT AUCTT 623 623-641 AUGCAAAUAAAACCG 645 AGUCCGGUUUUAUUUG 646 GACUTT CAUTT 624 624-642 UGCAAAUAAAACCGG 647 CAGUCCGGUUUUAUUU 648 ACUGTT GCATT 626 626-644 CAAAUAAAACCGGAC 649 UUCAGUCCGGUUUUAU 650 UGAATT UUGTT 627 627-645 AAAUAAAACCGGACU 651 CUUCAGUCCGGUUUUAU 652 GAAGTT UUTT 628 628-646 AAUAAAACCGGACUG 653 CCUUCAGUCCGGUUUUA 654 AAGGTT UUTT 630 630-648 UAAAACCGGACUGAA 655 CUCCUUCAGUCCGGUUU 656 GGAGTT UATT 631 631-649 AAAACCGGACUGAAG 657 GCUCCUUCAGUCCGGUU 658 GAGCTT UUTT 632 632-650 AAACCGGACUGAAGG 659 AGCUCCUUCAGUCCGGU 660 AGCUTT UUTT 633 633-651 AACCGGACUGAAGGA 661 CAGCUCCUUCAGUCCGG 662 GCUGTT UUTT 644 644-662 AGGAGCUGCCCAUGA 663 UUUCUCAUGGGCAGCUC 664 GAAATT CUTT 665 665-683 UACAGGAAAUCCUGC 665 CCAUGCAGGAUUUCCUG 666 AUGGTT UATT 668 668-686 AGGAAAUCCUGCAUG 667 GCGCCAUGCAGGAUUUC 668 GCGCTT CUTT 669 669-687 GGAAAUCCUGCAUGG 669 GGCGCCAUGCAGGAUUU 670 CGCCTT CCTT 670 670-688 GAAAUCCUGCAUGGC 671 CGGCGCCAUGCAGGAUU 672 GCCGTT UCTT 671 671-689 AAAUCCUGCAUGGCGC 673 ACGGCGCCAUGCAGGAU 674 CGUTT UUTT 672 672-690 AAUCCUGCAUGGCGCC 675 CACGGCGCCAUGCAGGA 676 GUGTT UUTT 674 674-692 UCCUGCAUGGCGCCGU 677 CGCACGGCGCCAUGCAG 678 GCGTT GATT 676 676-694 CUGCAUGGCGCCGUGC 679 ACCGCACGGCGCCAUGC 680 GGUTT AGTT 677 677-695 UGCAUGGCGCCGUGCG 681 AACCGCACGGCGCCAUG 682 GUUTT CATT 678 678-696 GCAUGGCGCCGUGCGG 683 GAACCGCACGGCGCCAU 684 UUCTT GCTT 680 680-698 AUGGCGCCGUGCGGU 685 CUGAACCGCACGGCGCC 686 UCAGTT AUTT 681 681-699 UGGCGCCGUGCGGUUC 687 GCUGAACCGCACGGCGC 688 AGCTT CATT 682 682-700 GGCGCCGUGCGGUUCA 689 UGCUGAACCGCACGGCG 690 GCATT CCTT 683 683-701 GCGCCGUGCGGUUCAG 691 UUGCUGAACCGCACGGC 692 CAATT GCTT 684 684-702 CGCCGUGCGGUUCAGC 693 GUUGCUGAACCGCACGG 694 AACTT CGTT 685 685-703 GCCGUGCGGUUCAGCA 695 UGUUGCUGAACCGCACG 696 ACATT GCTT 686 686-704 CCGUGCGGUUCAGCAA 697 UUGUUGCUGAACCGCAC 698 CAATT GGTT 688 688-706 GUGCGGUUCAGCAAC 699 GGUUGUUGCUGAACCGC 700 AACCTT ACTT 690 690-708 GCGGUUCAGCAACAAC 701 AGGGUUGUUGCUGAAC 702 CCUTT CGCTT 692 692-710 GGUUCAGCAACAACCC 703 GCAGGGUUGUUGCUGA 704 UGCTT ACCTT 698 698-716 GCAACAACCCUGCCCU 705 CACAGGGCAGGGUUGU 706 GUGTT UGCTT 700 700-718 AACAACCCUGCCCUGU 707 UGCACAGGGCAGGGUU 708 GCATT GUUTT 719 719-737 ACGUGGAGAGCAUCC 709 CACUGGAUGCUCUCCAC 710 AGUGTT GUTT 720 720-738 CGUGGAGAGCAUCCA 711 CCACUGGAUGCUCUCCA 712 GUGGTT CGTT 721 721-739 GUGGAGAGCAUCCAG 713 GCCACUGGAUGCUCUCC 714 UGGCTT ACTT 724 724-742 GAGAGCAUCCAGUGG 715 CCCGCCACUGGAUGCUC 716 CGGGTT UCTT 725 725-743 AGAGCAUCCAGUGGC 717 UCCCGCCACUGGAUGCU 718 GGGATT CUTT 726 726-744 GAGCAUCCAGUGGCG 719 GUCCCGCCACUGGAUGC 720 GGACTT UCTT 733 733-751 CAGUGGCGGGACAUA 721 UGACUAUGUCCCGCCAC 722 GUCATT UGTT 734 734-752 AGUGGCGGGACAUAG 723 CUGACUAUGUCCCGCCA 724 UCAGTT CUTT 736 736-754 UGGCGGGACAUAGUC 725 UGCUGACUAUGUCCCGC 726 AGCATT CATT 737 737-755 GGCGGGACAUAGUCA 727 CUGCUGACUAUGUCCCG 728 GCAGTT CCTT 763 763-781 CUCAGCAACAUGUCGA 729 CCAUCGACAUGUUGCUG 730 UGGTT AGTT 765 765-783 CAGCAACAUGUCGAU 731 GUCCAUCGACAUGUUGC 732 GGACTT UGTT 766 766-784 AGCAACAUGUCGAUG 733 AGUCCAUCGACAUGUUG 734 GACUTT CUTT 767 767-785 GCAACAUGUCGAUGG 735 AAGUCCAUCGACAUGUU 736 ACUUTT GCTT 769 769-787 AACAUGUCGAUGGAC 737 GGAAGUCCAUCGACAUG 738 UUCCTT UUTT 770 770-788 ACAUGUCGAUGGACU 739 UGGAAGUCCAUCGACAU 740 UCCATT GUTT 771 771-789 CAUGUCGAUGGACUU 741 CUGGAAGUCCAUCGACA 742 CCAGTT UGTT 772 772-790 AUGUCGAUGGACUUC 743 UCUGGAAGUCCAUCGAC 744 CAGATT AUTT 775 775-793 UCGAUGGACUUCCAG 745 GGUUCUGGAAGUCCAUC 746 AACCTT GATT 789 789-807 GAACCACCUGGGCAGC 747 GCAGCUGCCCAGGUGGU 748 UGCTT UCTT 798 798-816 GGGCAGCUGCCAAAA 749 ACACUUUUGGCAGCUGC 750 GUGUTT CCTT 800 800-818 GCAGCUGCCAAAAGU 751 UCACACUUUUGGCAGCU 752 GUGATT GCTT 805 805-823 UGCCAAAAGUGUGAU 753 UUGGAUCACACUUUUG 754 CCAATT GCATT 806 806-824 GCCAAAAGUGUGAUC 755 CUUGGAUCACACUUUUG 756 CAAGTT GCTT 807 807-825 CCAAAAGUGUGAUCC 757 GCUUGGAUCACACUUUU 758 AAGCTT GGTT 810 810-828 AAAGUGUGAUCCAAG 759 ACAGCUUGGAUCACACU 760 CUGUTT UUTT 814 814-832 UGUGAUCCAAGCUGU 761 UGGGACAGCUUGGAUC 762 CCCATT ACATT 815 815-833 GUGAUCCAAGCUGUCC 763 UUGGGACAGCUUGGAU 764 CAATT CACTT 817 817-835 GAUCCAAGCUGUCCCA 765 CAUUGGGACAGCUUGG 766 AUGTT AUCTT 818 818-836 AUCCAAGCUGUCCCAA 767 CCAUUGGGACAGCUUGG 768 UGGTT AUTT 819 819-837 UCCAAGCUGUCCCAAU 769 CCCAUUGGGACAGCUUG 770 GGGTT GATT 820 820-838 CCAAGCUGUCCCAAUG 771 UCCCAUUGGGACAGCUU 772 GGATT GGTT 821 821-839 CAAGCUGUCCCAAUGG 773 CUCCCAUUGGGACAGCU 774 GAGTT UGTT 823 823-841 AGCUGUCCCAAUGGG 775 AGCUCCCAUUGGGACAG 776 AGCUTT CUTT 826 826-844 UGUCCCAAUGGGAGC 777 AGCAGCUCCCAUUGGGA 778 UGCUTT CATT 847 847-865 GGUGCAGGAGAGGAG 779 AGUUCUCCUCUCCUGCA 780 AACUTT CCTT 871 871-889 AAACUGACCAAAAUC 781 AGAUGAUUUUGGUCAG 782 AUCUTT UUUTT 872 872-890 AACUGACCAAAAUCA 783 CAGAUGAUUUUGGUCA 784 UCUGTT GUUTT 873 873-891 ACUGACCAAAAUCAUC 785 ACAGAUGAUUUUGGUC 786 UGUTT AGUTT 877 877-895 ACCAAAAUCAUCUGU 787 GGGCACAGAUGAUUUU 788 GCCCTT GGUTT 878 878-896 CCAAAAUCAUCUGUGC 789 UGGGCACAGAUGAUUU 790 CCATT UGGTT 881 881-899 AAAUCAUCUGUGCCCA 791 UGCUGGGCACAGAUGA 792 GCATT UUUTT 890 890-908 GUGCCCAGCAGUGCUC 793 CCGGAGCACUGCUGGGC 794 CGGTT ACTT 892 892-910 GCCCAGCAGUGCUCCG 795 GCCCGGAGCACUGCUGG 796 GGCTT GCTT 929 929-947 CCAGUGACUGCUGCCA 797 UUGUGGCAGCAGUCACU 798 CAATT GGTT 930 930-948 CAGUGACUGCUGCCAC 799 GUUGUGGCAGCAGUCAC 800 AACTT UGTT 979 979-997 GAGAGCGACUGCCUG 801 AGACCAGGCAGUCGCUC 802 GUCUTT UCTT 980 980-998 AGAGCGACUGCCUGG 803 CAGACCAGGCAGUCGCU 804 UCUGTT CUTT 981 981-999 GAGCGACUGCCUGGUC 805 GCAGACCAGGCAGUCGC 806 UGCTT UCTT 982  982-1000 AGCGACUGCCUGGUCU 807 GGCAGACCAGGCAGUCG 808 GCCTT CUTT 983  983-1001 GCGACUGCCUGGUCUG 809 CGGCAGACCAGGCAGUC 810 CCGTT GCTT 984  984-1002 CGACUGCCUGGUCUGC 811 GCGGCAGACCAGGCAGU 812 CGCTT CGTT 989  989-1007 GCCUGGUCUGCCGCAA 813 AAUUUGCGGCAGACCAG 814 AUUTT GCTT 990  990-1008 CCUGGUCUGCCGCAAA 815 GAAUUUGCGGCAGACCA 816 UUCTT GGTT 991  991-1009 CUGGUCUGCCGCAAAU 817 GGAAUUUGCGGCAGACC 818 UCCTT AGTT 992  992-1010 UGGUCUGCCGCAAAU 819 CGGAAUUUGCGGCAGAC 820 UCCGTT CATT 994  994-1012 GUCUGCCGCAAAUUCC 821 CUCGGAAUUUGCGGCAG 822 GAGTT ACTT 995  995-1013 UCUGCCGCAAAUUCCG 823 UCUCGGAAUUUGCGGCA 824 AGATT GATT 996  996-1014 CUGCCGCAAAUUCCGA 825 GUCUCGGAAUUUGCGGC 826 GACTT AGTT 997  997-1015 UGCCGCAAAUUCCGAG 827 CGUCUCGGAAUUUGCGG 828 ACGTT CATT 999  999-1017 CCGCAAAUUCCGAGAC 829 UUCGUCUCGGAAUUUGC 830 GAATT GGTT 1004 1004-1022 AAUUCCGAGACGAAG 831 GUGGCUUCGUCUCGGAA 832 CCACTT UUTT 1005 1005-1023 AUUCCGAGACGAAGCC 833 CGUGGCUUCGUCUCGGA 834 ACGTT AUTT 1006 1006-1024 UUCCGAGACGAAGCCA 835 ACGUGGCUUCGUCUCGG 836 CGUTT AATT 1007 1007-1025 UCCGAGACGAAGCCAC 837 CACGUGGCUUCGUCUCG 838 GUGTT GATT 1008 1008-1026 CCGAGACGAAGCCACG 839 GCACGUGGCUUCGUCUC 840 UGCTT GGTT 1010 1010-1028 GAGACGAAGCCACGU 841 UUGCACGUGGCUUCGUC 842 GCAATT UCTT 1013 1013-1031 ACGAAGCCACGUGCAA 843 UCCUUGCACGUGGCUUC 844 GGATT GUTT 1014 1014-1032 CGAAGCCACGUGCAAG 845 GUCCUUGCACGUGGCUU 846 GACTT CGTT 1015 1015-1033 GAAGCCACGUGCAAG 847 UGUCCUUGCACGUGGCU 848 GACATT UCTT 1016 1016-1034 AAGCCACGUGCAAGG 849 GUGUCCUUGCACGUGGC 850 ACACTT UUTT 1040 1040-1058 CCCCACUCAUGCUCUA 851 UUGUAGAGCAUGAGUG 852 CAATT GGGTT 1042 1042-1060 CCACUCAUGCUCUACA 853 GGUUGUAGAGCAUGAG 854 ACCTT UGGTT 1044 1044-1062 ACUCAUGCUCUACAAC 855 GGGGUUGUAGAGCAUG 856 CCCTT AGUTT 1047 1047-1065 CAUGCUCUACAACCCC 857 GGUGGGGUUGUAGAGC 858 ACCTT AUGTT 1071 1071-1089 CCAGAUGGAUGUGAA 859 GGGGUUCACAUCCAUCU 860 CCCCTT GGTT 1073 1073-1091 AGAUGGAUGUGAACC 861 UCGGGGUUCACAUCCAU 862 CCGATT CUTT 1074 1074-1092 GAUGGAUGUGAACCC 863 CUCGGGGUUCACAUCCA 864 CGAGTT UCTT 1075 1075-1093 AUGGAUGUGAACCCC 865 CCUCGGGGUUCACAUCC 866 GAGGTT AUTT 1077 1077-1095 GGAUGUGAACCCCGA 867 GCCCUCGGGGUUCACAU 868 GGGCTT CCTT 1078 1078-1096 GAUGUGAACCCCGAG 869 UGCCCUCGGGGUUCACA 870 GGCATT UCTT 1080 1080-1098 UGUGAACCCCGAGGGC 871 UUUGCCCUCGGGGUUCA 872 AAATT CATT 1084 1084-1102 AACCCCGAGGGCAAAU 873 UGUAUUUGCCCUCGGGG 874 ACATT UUTT 1085 1085-1103 ACCCCGAGGGCAAAUA 875 CUGUAUUUGCCCUCGGG 876 CAGTT GUTT 1087 1087-1105 CCCGAGGGCAAAUACA 877 AGCUGUAUUUGCCCUCG 878 GCUTT GGTT 1088 1088-1106 CCGAGGGCAAAUACA 879 AAGCUGUAUUUGCCCUC 880 GCUUTT GGTT 1089 1089-1107 CGAGGGCAAAUACAG 881 AAAGCUGUAUUUGCCCU 882 CUUUTT CGTT 1096 1096-1114 AAAUACAGCUUUGGU 883 UGGCACCAAAGCUGUAU 884 GCCATT UUTT 1097 1097-1115 AAUACAGCUUUGGUG 885 GUGGCACCAAAGCUGUA 886 CCACTT UUTT 1098 1098-1116 AUACAGCUUUGGUGC 887 GGUGGCACCAAAGCUGU 888 CACCTT AUTT 1104 1104-1122 CUUUGGUGCCACCUGC 889 CACGCAGGUGGCACCAA 890 GUGTT AGTT 1106 1106-1124 UUGGUGCCACCUGCGU 891 UUCACGCAGGUGGCACC 892 GAATT AATT 1112 1112-1130 CCACCUGCGUGAAGAA 893 CACUUCUUCACGCAGGU 894 GUGTT GGTT 1116 1116-1134 CUGCGUGAAGAAGUG 895 GGGACACUUCUUCACGC 896 UCCCTT AGTT 1117 1117-1135 UGCGUGAAGAAGUGU 897 GGGGACACUUCUUCACG 898 CCCCTT CATT 1118 1118-1136 GCGUGAAGAAGUGUC 899 CGGGGACACUUCUUCAC 900 CCCGTT GCTT 1119 1119-1137 CGUGAAGAAGUGUCC 901 ACGGGGACACUUCUUCA 902 CCGUTT CGTT 1120 1120-1138 GUGAAGAAGUGUCCC 903 UACGGGGACACUUCUUC 904 CGUATT ACTT 1121 1121-1139 UGAAGAAGUGUCCCC 905 UUACGGGGACACUUCUU 906 GUAATT CATT 1122 1122-1140 GAAGAAGUGUCCCCG 907 AUUACGGGGACACUUCU 908 UAAUTT UCTT 1123 1123-1141 AAGAAGUGUCCCCGU 909 AAUUACGGGGACACUUC 910 AAUUTT UUTT 1124 1124-1142 AGAAGUGUCCCCGUA 911 UAAUUACGGGGACACU 912 AUUATT UCUTT 1125 1125-1143 GAAGUGUCCCCGUAA 913 AUAAUUACGGGGACAC 914 UUAUTT UUCTT 1126 1126-1144 AAGUGUCCCCGUAAU 915 CAUAAUUACGGGGACAC 916 UAUGTT UUTT 1127 1127-1145 AGUGUCCCCGUAAUU 917 ACAUAAUUACGGGGAC 918 AUGUTT ACUTT 1128 1128-1146 GUGUCCCCGUAAUUA 919 CACAUAAUUACGGGGAC 920 UGUGTT ACTT 1129 1129-1147 UGUCCCCGUAAUUAU 921 CCACAUAAUUACGGGGA 922 GUGGTT CATT 1130 1130-1148 GUCCCCGUAAUUAUG 923 ACCACAUAAUUACGGGG 924 UGGUTT ACTT 1132 1132-1150 CCCCGUAAUUAUGUG 925 UCACCACAUAAUUACGG 926 GUGATT GGTT 1134 1134-1152 CCGUAAUUAUGUGGU 927 UGUCACCACAUAAUUAC 928 GACATT GGTT 1136 1136-1154 GUAAUUAUGUGGUGA 929 UCUGUCACCACAUAAUU 930 CAGATT ACTT 1137 1137-1155 UAAUUAUGUGGUGAC 931 AUCUGUCACCACAUAAU 932 AGAUTT UATT 1138 1138-1156 AAUUAUGUGGUGACA 933 GAUCUGUCACCACAUAA 934 GAUCTT UUTT 1139 1139-1157 AUUAUGUGGUGACAG 935 UGAUCUGUCACCACAUA 936 AUCATT AUTT 1140 1140-1158 UUAUGUGGUGACAGA 937 GUGAUCUGUCACCACAU 938 UCACTT AATT 1142 1142-1160 AUGUGGUGACAGAUC 939 CCGUGAUCUGUCACCAC 940 ACGGTT AUTT 1145 1145-1163 UGGUGACAGAUCACG 941 GAGCCGUGAUCUGUCAC 942 GCUCTT CATT 1147 1147-1165 GUGACAGAUCACGGC 943 ACGAGCCGUGAUCUGUC 944 UCGUTT ACTT 1148 1148-1166 UGACAGAUCACGGCUC 945 CACGAGCCGUGAUCUGU 946 GUGTT CATT 1149 1149-1167 GACAGAUCACGGCUCG 947 GCACGAGCCGUGAUCUG 948 UGCTT UCTT 1150 1150-1168 ACAGAUCACGGCUCGU 949 CGCACGAGCCGUGAUCU 950 GCGTT GUTT 1151 1151-1169 CAGAUCACGGCUCGUG 951 ACGCACGAGCCGUGAUC 952 CGUTT UGTT 1152 1152-1170 AGAUCACGGCUCGUGC 953 GACGCACGAGCCGUGAU 954 GUCTT CUTT 1153 1153-1171 GAUCACGGCUCGUGCG 955 GGACGCACGAGCCGUGA 956 UCCTT UCTT 1154 1154-1172 AUCACGGCUCGUGCGU 957 CGGACGCACGAGCCGUG 958 CCGTT AUTT 1155 1155-1173 UCACGGCUCGUGCGUC 959 UCGGACGCACGAGCCGU 960 CGATT GATT 1156 1156-1174 CACGGCUCGUGCGUCC 961 CUCGGACGCACGAGCCG 962 GAGTT UGTT 1157 1157-1175 ACGGCUCGUGCGUCCG 963 GCUCGGACGCACGAGCC 964 AGCTT GUTT 1160 1160-1178 GCUCGUGCGUCCGAGC 965 CAGGCUCGGACGCACGA 966 CUGTT GCTT 1200 1200-1218 GGAGGAAGACGGCGU 967 GCGGACGCCGUCUUCCU 968 CCGCTT CCTT 1201 1201-1219 GAGGAAGACGGCGUC 969 UGCGGACGCCGUCUUCC 970 CGCATT UCTT 1203 1203-1221 GGAAGACGGCGUCCGC 971 CUUGCGGACGCCGUCUU 972 AAGTT CCTT 1204 1204-1222 GAAGACGGCGUCCGCA 973 ACUUGCGGACGCCGUCU 974 AGUTT UCTT 1205 1205-1223 AAGACGGCGUCCGCAA 975 CACUUGCGGACGCCGUC 976 GUGTT UUTT 1207 1207-1225 GACGGCGUCCGCAAGU 977 UACACUUGCGGACGCCG 978 GUATT UCTT 1208 1208-1226 ACGGCGUCCGCAAGUG 979 UUACACUUGCGGACGCC 980 UAATT GUTT 1211 1211-1229 GCGUCCGCAAGUGUA 981 UUCUUACACUUGCGGAC 982 AGAATT GCTT 1212 1212-1230 CGUCCGCAAGUGUAA 983 CUUCUUACACUUGCGGA 984 GAAGTT CGTT 1213 1213-1231 GUCCGCAAGUGUAAG 985 ACUUCUUACACUUGCGG 986 AAGUTT ACTT 1214 1214-1232 UCCGCAAGUGUAAGA 987 CACUUCUUACACUUGCG 988 AGUGTT GATT 1215 1215-1233 CCGCAAGUGUAAGAA 989 GCACUUCUUACACUUGC 990 GUGCTT GGTT 1216 1216-1234 CGCAAGUGUAAGAAG 991 CGCACUUCUUACACUUG 992 UGCGTT CGTT 1217 1217-1235 GCAAGUGUAAGAAGU 993 UCGCACUUCUUACACUU 994 GCGATT GCTT 1219 1219-1237 AAGUGUAAGAAGUGC 995 CUUCGCACUUCUUACAC 996 GAAGTT UUTT 1220 1220-1238 AGUGUAAGAAGUGCG 997 CCUUCGCACUUCUUACA 998 AAGGTT CUTT 1221 1221-1239 GUGUAAGAAGUGCGA 999 CCCUUCGCACUUCUUAC 1000 AGGGTT ACTT 1222 1222-1240 UGUAAGAAGUGCGAA 1001 GCCCUUCGCACUUCUUA 1002 GGGCTT CATT 1223 1223-1241 GUAAGAAGUGCGAAG 1003 GGCCCUUCGCACUUCUU 1004 GGCCTT ACTT 1224 1224-1242 UAAGAAGUGCGAAGG 1005 AGGCCCUUCGCACUUCU 1006 GCCUTT UATT 1225 1225-1243 AAGAAGUGCGAAGGG 1007 AAGGCCCUUCGCACUUC 1008 CCUUTT UUTT 1226 1226-1244 AGAAGUGCGAAGGGC 1009 CAAGGCCCUUCGCACUU 1010 CUUGTT CUTT 1229 1229-1247 AGUGCGAAGGGCCUU 1011 CGGCAAGGCCCUUCGCA 1012 GCCGTT CUTT 1230 1230-1248 GUGCGAAGGGCCUUG 1013 GCGGCAAGGCCCUUCGC 1014 CCGCTT ACTT 1231 1231-1249 UGCGAAGGGCCUUGCC 1015 UGCGGCAAGGCCCUUCG 1016 GCATT CATT 1232 1232-1250 GCGAAGGGCCUUGCCG 1017 UUGCGGCAAGGCCCUUC 1018 CAATT GCTT 1233 1233-1251 CGAAGGGCCUUGCCGC 1019 UUUGCGGCAAGGCCCUU 1020 AAATT CGTT 1235 1235-1253 AAGGGCCUUGCCGCAA 1021 ACUUUGCGGCAAGGCCC 1022 AGUTT UUTT 1236 1236-1254 AGGGCCUUGCCGCAAA 1023 CACUUUGCGGCAAGGCC 1024 GUGTT CUTT 1237 1237-1255 GGGCCUUGCCGCAAAG 1025 ACACUUUGCGGCAAGGC 1026 UGUTT CCTT 1238 1238-1256 GGCCUUGCCGCAAAGU 1027 CACACUUUGCGGCAAGG 1028 GUGTT CCTT 1239 1239-1257 GCCUUGCCGCAAAGUG 1029 ACACACUUUGCGGCAAG 1030 UGUTT GCTT 1241 1241-1259 CUUGCCGCAAAGUGU 1031 UUACACACUUUGCGGCA 1032 GUAATT AGTT 1261 1261-1279 GGAAUAGGUAUUGGU 1033 AUUCACCAAUACCUAUU 1034 GAAUTT CCTT 1262 1262-1280 GAAUAGGUAUUGGUG 1035 AAUUCACCAAUACCUAU 1036 AAUUTT UCTT 1263 1263-1281 AAUAGGUAUUGGUGA 1037 AAAUUCACCAAUACCUA 1038 AUUUTT UUTT 1264 1264-1282 AUAGGUAUUGGUGAA 1039 UAAAUUCACCAAUACCU 1040 UUUATT AUTT 1266 1266-1284 AGGUAUUGGUGAAUU 1041 UUUAAAUUCACCAAUAC 1042 UAAATT CUTT 1267 1267-1285 GGUAUUGGUGAAUUU 1043 CUUUAAAUUCACCAAUA 1044 AAAGTT CCTT 1289 1289-1307 CACUCUCCAUAAAUGC 1045 GUAGCAUUUAUGGAGA 1046 UACTT GUGTT 1313 1313-1331 UUAAACACUUCAAAA 1047 CAGUUUUUGAAGUGUU 1048 ACUGTT UAATT 1320 1320-1338 CUUCAAAAACUGCACC 1049 GGAGGUGCAGUUUUUG 1050 UCCTT AAGTT 1321 1321-1339 UUCAAAAACUGCACCU 1051 UGGAGGUGCAGUUUUU 1052 CCATT GAATT 1322 1322-1340 UCAAAAACUGCACCUC 1053 AUGGAGGUGCAGUUUU 1054 CAUTT UGATT 1323 1323-1341 CAAAAACUGCACCUCC 1055 GAUGGAGGUGCAGUUU 1056 AUCTT UUGTT 1324 1324-1342 AAAAACUGCACCUCCA 1057 UGAUGGAGGUGCAGUU 1058 UCATT UUUTT 1328 1328-1346 ACUGCACCUCCAUCAG 1059 CCACUGAUGGAGGUGCA 1060 UGGTT GUTT 1332 1332-1350 CACCUCCAUCAGUGGC 1061 AUCGCCACUGAUGGAGG 1062 GAUTT UGTT 1333 1333-1351 ACCUCCAUCAGUGGCG 1063 GAUCGCCACUGAUGGAG 1064 AUCTT GUTT 1335 1335-1353 CUCCAUCAGUGGCGAU 1065 GAGAUCGCCACUGAUGG 1066 CUCTT AGTT 1338 1338-1356 CAUCAGUGGCGAUCUC 1067 GUGGAGAUCGCCACUGA 1068 CACTT UGTT 1344 1344-1362 UGGCGAUCUCCACAUC 1069 CAGGAUGUGGAGAUCG 1070 CUGTT CCATT 1345 1345-1363 GGCGAUCUCCACAUCC 1071 GCAGGAUGUGGAGAUC 1072 UGCTT GCCTT 1346 1346-1364 GCGAUCUCCACAUCCU 1073 GGCAGGAUGUGGAGAU 1074 GCCTT CGCTT 1347 1347-1365 CGAUCUCCACAUCCUG 1075 CGGCAGGAUGUGGAGA 1076 CCGTT UCGTT 1348 1348-1366 GAUCUCCACAUCCUGC 1077 CCGGCAGGAUGUGGAG 1078 CGGTT AUCTT 1353 1353-1371 CCACAUCCUGCCGGUG 1079 UGCCACCGGCAGGAUGU 1080 GCATT GGTT 1354 1354-1372 CACAUCCUGCCGGUGG 1081 AUGCCACCGGCAGGAUG 1082 CAUTT UGTT 1355 1355-1373 ACAUCCUGCCGGUGGC 1083 AAUGCCACCGGCAGGAU 1084 AUUTT GUTT 1357 1357-1375 AUCCUGCCGGUGGCAU 1085 UAAAUGCCACCGGCAGG 1086 UUATT AUTT 1360 1360-1378 CUGCCGGUGGCAUUU 1087 CCCUAAAUGCCACCGGC 1088 AGGGTT AGTT 1361 1361-1379 UGCCGGUGGCAUUUA 1089 CCCCUAAAUGCCACCGG 1090 GGGGTT CATT 1362 1362-1380 GCCGGUGGCAUUUAG 1091 ACCCCUAAAUGCCACCG 1092 GGGUTT GCTT 1363 1363-1381 CCGGUGGCAUUUAGG 1093 CACCCCUAAAUGCCACC 1094 GGUGTT GGTT 1366 1366-1384 GUGGCAUUUAGGGGU 1095 AGUCACCCCUAAAUGCC 1096 GACUTT ACTT 1369 1369-1387 GCAUUUAGGGGUGAC 1097 AGGAGUCACCCCUAAAU 1098 UCCUTT GCTT 1370 1370-1388 CAUUUAGGGGUGACU 1099 AAGGAGUCACCCCUAAA 1100 CCUUTT UGTT 1371 1371-1389 AUUUAGGGGUGACUC 1101 GAAGGAGUCACCCCUAA 1102 CUUCTT AUTT 1372 1372-1390 UUUAGGGGUGACUCC 1103 UGAAGGAGUCACCCCUA 1104 UUCATT AATT 1373 1373-1391 UUAGGGGUGACUCCU 1105 GUGAAGGAGUCACCCCU 1106 UCACTT AATT 1374 1374-1392 UAGGGGUGACUCCUU 1107 UGUGAAGGAGUCACCCC 1108 CACATT UATT 1404 1404-1422 UCUGGAUCCACAGGA 1109 CAGUUCCUGUGGAUCCA 1110 ACUGTT GATT 1408 1408-1426 GAUCCACAGGAACUG 1111 UAUCCAGUUCCUGUGGA 1112 GAUATT UCTT 1409 1409-1427 AUCCACAGGAACUGG 1113 AUAUCCAGUUCCUGUGG 1114 AUAUTT AUTT 1411 1411-1429 CCACAGGAACUGGAU 1115 GAAUAUCCAGUUCCUGU 1116 AUUCTT GGTT 1412 1412-1430 CACAGGAACUGGAUA 1117 AGAAUAUCCAGUUCCUG 1118 UUCUTT UGTT 1419 1419-1437 ACUGGAUAUUCUGAA 1119 GGUUUUCAGAAUAUCC 1120 AACCTT AGUTT 1426 1426-1444 AUUCUGAAAACCGUA 1121 CCUUUACGGUUUUCAGA 1122 AAGGTT AUTT 1427 1427-1445 UUCUGAAAACCGUAA 1123 UCCUUUACGGUUUUCAG 1124 AGGATT AATT 1430 1430-1448 UGAAAACCGUAAAGG 1125 AUUUCCUUUACGGUUU 1126 AAAUTT UCATT 1431 1431-1449 GAAAACCGUAAAGGA 1127 GAUUUCCUUUACGGUU 1128 AAUCTT UUCTT

TABLE 3 EGFR siRNA Sequences with Chemical Modifications Sequence SEQ SEQ position in sense strand sequence (5′- ID antisense strand sequence ID hs Id # NM_005228.3 3′) NO: (5′-3′) NO: 68 68-86 cgGfcCfgGfaGfuCfcCfgAf 1129 UfAfgCfuCfgGfgAfcUfcCfgG 1130 gCfuAfdTsdT fcCfgdTsdT 71 71-89 ccGfgAfgUfcCfcGfaGfcUf 1131 GfGfcUfaGfcUfcGfgGfaCfuC 1132 aGfcCfdTsdT fcGfgdTsdT 72 72-90 cgGfaGfuCfcCfgAfgCfuAf 1133 GfGfgCfuAfgCfuCfgGfgAfcU 1134 gCfcCfdTsdT fcCfgdTsdT 73 73-91 ggAfgUfcCfcGfaGfcUfaGf 1135 GfGfgGfcUfaGfcUfcGfgGfaC 1136 cCfcCfdTsdT fuCfcdTsdT 74 74-92 gaGfuCfcCfgAfgCfuAfgCf 1137 CfGfgGfgCfuAfgCfuCfgGfgA 1138 cCfcGfdTsdT fcUfcdTsdT 75 75-93 agUfcCfcGfaGfcUfaGfcCf 1139 CfCfgGfgGfcUfaGfcUfcGfgG 1140 cCfgGfdTsdT faCfudTsdT 76 76-94 guCfcCfgAfgCfuAfgCfcCf 1141 GfCfcGfgGfgCfuAfgCfuCfgG 1142 cGfgCfdTsdT fgAfcdTsdT 78 78-96 ccCfgAfgCfuAfgCfcCfcGf 1143 CfCfgCfcGfgGfgCfuAfgCfuC 1144 gCfgGfdTsdT fgGfgdTsdT 114 114-132 ggAfcGfaCfaGfgCfcAfcCf 1145 AfCfgAfgGfuGfgCfcUfgUfcG 1146 uCfgUfdTsdT fuCfcdTsdT 115 115-133 gaCfgAfcAfgGfcCfaCfcUf 1147 GfAfcGfaGfgUfgGfcCfuGfuC 1148 cGfuCfdTsdT fgUfcdTsdT 116 116-134 acGfaCfaGfgCfcAfcCfuCf 1149 CfGfaCfgAfgGfuGfgCfcUfgU 1150 gUfcGfdTsdT fcGfudTsdT 117 117-135 cgAfcAfgGfcCfaCfcUfcGf 1151 CfCfgAfcGfaGfgUfgGfcCfuG 1152 uCfgGfdTsdT fuCfgdTsdT 118 118-136 gaCfaGfgCfcAfcCfuCfgUf 1153 GfCfcGfaCfgAfgGfuGfgCfcU 1154 cGfgCfdTsdT fgUfcdTsdT 120 120-138 caGfgCfcAfcCfuCfgUfcGf 1155 AfCfgCfcGfaCfgAfgGfuGfgC 1156 gCfgUfdTsdT fcUfgdTsdT 121 121-139 agGfcCfaCfcUfcGfuCfgGf 1157 GfAfcGfcCfgAfcGfaGfgUfgG 1158 cGfuCfdTsdT fcCfudTsdT 122 122-140 ggCfcAfcCfuCfgUfcGfgCf 1159 GfGfaCfgCfcGfaCfgAfgGfuG 1160 gUfcCfdTsdT fgCfcdTsdT 123 123-141 gcCfaCfcUfcGfuCfgGfcGf 1161 CfGfgAfcGfcCfgAfcGfaGfgU 1162 uCfcGfdTsdT fgGfcdTsdT 124 124-142 ccAfcCfuCfgUfcGfgCfgUf 1163 GfCfgGfaCfgCfcGfaCfgAfgG 1164 cCfgCfdTsdT fuGfgdTsdT 125 125-143 caCfcUfcGfuCfgGfcGfuCf 1165 GfGfcGfgAfcGfcCfgAfcGfaG 1166 cGfcCfdTsdT fgUfgdTsdT 126 126-144 acCfuCfgUfcGfgCfgUfcCf 1167 GfGfgCfgGfaCfgCfcGfaCfgA 1168 gCfcCfdTsdT fgGfudTsdT 127 127-145 ccUfcGfuCfgGfcGfuCfcGf 1169 CfGfgGfcGfgAfcGfcCfgAfcG 1170 cCfcGfdTsdT faGfgdTsdT 128 128-146 cuCfgUfcGfgCfgUfcCfgCf 1171 UfCfgGfgCfgGfaCfgCfcGfaC 1172 cCfgAfdTsdT fgAfgdTsdT 129 129-147 ucGfuCfgGfcGfuCfcGfcCf 1173 CfUfcGfgGfcGfgAfcGfcCfgA 1174 cGfaGfdTsdT fcGfadTsdT 130 130-148 cgUfcGfgCfgUfcCfgCfcCf 1175 AfCfuCfgGfgCfgGfaCfgCfcG 1176 gAfgUfdTsdT faCfgdTsdT 131 131-149 guCfgGfcGfuCfcGfcCfcGf 1177 GfAfcUfcGfgGfcGfgAfcGfcC 1178 aGfuCfdTsdT fgAfcdTsdT 132 132-150 ucGfgCfgUfcCfgCfcCfgAf 1179 GfGfaCfuCfgGfgCfgGfaCfgC 1180 gUfcCfdTsdT fcGfadTsdT 135 135-153 gcGfuCfcGfcCfcGfaGfuCf 1181 CfGfgGfgAfcUfcGfgGfcGfgA 1182 cCfcGfdTsdT fcGfcdTsdT 136 136-154 cgUfcCfgCfcCfgAfgUfcCf 1183 GfCfgGfgGfaCfuCfgGfgCfgG 1184 cCfgCfdTsdT faCfgdTsdT 141 141-159 gcCfcGfaGfuCfcCfcGfcCf 1185 GfCfgAfgGfcGfgGfgAfcUfcG 1186 uCfgCfdTsdT fgGfcdTsdT 164 164-182 aaCfgCfcAfcAfaCfcAfcCfg 1187 GfCfgCfgGfuGfgUfuGfuGfgC 1188 CfgCfdTsdT fgUfudTsdT 165 165-183 acGfcCfaCfaAfcCfaCfcGfc 1189 UfGfcGfcGfgUfgGfuUfgUfg 1190 GfcAfdTsdT GfcGfudTsdT 166 166-184 cgCfcAfcAfaCfcAfcCfgCf 1191 GfUfgCfgCfgGfuGfgUfuGfu 1192 gCfaCfdTsdT GfgCfgdTsdT 168 168-186 ccAfcAfaCfcAfcCfgCfgCfa 1193 CfCfgUfgCfgCfgGfuGfgUfuG 1194 CfgGfdTsdT fuGfgdTsdT 169 169-187 caCfaAfcCfaCfcGfcGfcAfc 1195 GfCfcGfuGfcGfcGfgUfgGfuU 1196 GfgCfdTsdT fgUfgdTsdT 170 170-188 acAfaCfcAfcCfgCfgCfaCfg 1197 GfGfcCfgUfgCfgCfgGfuGfgU 1198 GfcCfdTsdT fuGfudTsdT 247 247-265 auGfcGfaCfcCfuCfcGfgGf 1199 CfCfgUfcCfcGfgAfgGfgUfcG 1200 aCfgGfdTsdT fcAfudTsdT 248 248-266 ugCfgAfcCfcUfcCfgGfgAf 1201 GfCfcGfuCfcCfgGfaGfgGfuC 1202 cGfgCfdTsdT fgCfadTsdT 249 249-267 gcGfaCfcCfuCfcGfgGfaCf 1203 GfGfcCfgUfcCfcGfgAfgGfgU 1204 gGfcCfdTsdT fcGfcdTsdT 251 251-269 gaCfcCfuCfcGfgGfaCfgGf 1205 CfCfgGfcCfgUfcCfcGfgAfgG 1206 cCfgGfdTsdT fgUfcdTsdT 252 252-270 acCfcUfcCfgGfgAfcGfgCf 1207 CfCfcGfgCfcGfuCfcCfgGfaGf 1208 cGfgGfdTsdT gGfudTsdT 254 254-272 ccUfcCfgGfgAfcGfgCfcGf 1209 GfCfcCfcGfgCfcGfuCfcCfgGf 1210 gGfgCfdTsdT aGfgdTsdT 329 329-347 agAfaAfgUfuUfgCfcAfaGf 1211 GfUfgCfcUfuGfgCfaAfaCfuU 1212 gCfaCfdTsdT fuCfudTsdT 330 330-348 gaAfaGfuUfuGfcCfaAfgGf 1213 CfGfuGfcCfuUfgGfcAfaAfcU 1214 cAfcGfdTsdT fuUfcdTsdT 332 332-350 aaGfuUfuGfcCfaAfgGfcAf 1215 CfUfcGfuGfcCfuUfgGfcAfaA 1216 cGfaGfdTsdT fcUfudTsdT 333 333-351 agUfuUfgCfcAfaGfgCfaCf 1217 AfCfuCfgUfgCfcUfuGfgCfaA 1218 gAfgUfdTsdT faCfudTsdT 334 334-352 guUfuGfcCfaAfgGfcAfcGf 1219 UfAfcUfcGfuGfcCfuUfgGfcA 1220 aGfuAfdTsdT faAfcdTsdT 335 335-353 uuUfgCfcAfaGfgCfaCfgAf 1221 UfUfaCfuCfgUfgCfcUfuGfgC 1222 gUfaAfdTsdT faAfadTsdT 336 336-354 uuGfcCfaAfgGfcAfcGfaGf 1223 GfUfuAfcUfcGfuGfcCfuUfgG 1224 uAfaCfdTsdT fcAfadTsdT 337 337-355 ugCfcAfaGfgCfaCfgAfgUf 1225 UfGfuUfaCfuCfgUfgCfcUfuG 1226 aAfcAfdTsdT fgCfadTsdT 338 338-356 gcCfaAfgGfcAfcGfaGfuAf 1227 UfUfgUfuAfcUfcGfuGfcCfuU 1228 aCfaAfdTsdT fgGfcdTsdT 361 361-379 acGfcAfgUfuGfgGfcAfcUf 1229 CfAfaAfaGfuGfcCfcAfaCfuG 1230 uUfuGfdTsdT fcGfudTsdT 362 362-380 cgCfaGfuUfgGfgCfaCfuUf 1231 UfCfaAfaAfgUfgCfcCfaAfcU 1232 uUfgAfdTsdT fgCfgdTsdT 363 363-381 gcAfgUfuGfgGfcAfcUfuUf 1233 UfUfcAfaAfaGfuGfcCfcAfaC 1234 uGfaAfdTsdT fuGfcdTsdT 364 364-382 caGfuUfgGfgCfaCfuUfuUf 1235 CfUfuCfaAfaAfgUfgCfcCfaA 1236 gAfaGfdTsdT fcUfgdTsdT 365 365-383 agUfuGfgGfcAfcUfuUfuGf 1237 UfCfuUfcAfaAfaGfuGfcCfcA 1238 aAfgAfdTsdT faCfudTsdT 366 366-384 guUfgGfgCfaCfuUfuUfgAf 1239 AfUfcUfuCfaAfaAfgUfgCfcC 1240 aGfaUfdTsdT faAfcdTsdT 367 367-385 uuGfgGfcAfcUfuUfuGfaAf 1241 GfAfuCfuUfcAfaAfaGfuGfcC 1242 gAfuCfdTsdT fcAfadTsdT 368 368-386 ugGfgCfaCfuUfuUfgAfaGf 1243 UfGfaUfcUfuCfaAfaAfgUfgC 1244 aUfcAfdTsdT fcCfadTsdT 369 369-387 ggGfcAfcUfuUfuGfaAfgAf 1245 AfUfgAfuCfuUfcAfaAfaGfuG 1246 uCfaUfdTsdT fcCfcdTsdT 377 377-395 uuGfaAfgAfuCfaUfuUfuCf 1247 CfUfgAfgAfaAfaUfgAfuCfuU 1248 uCfaGfdTsdT fcAfadTsdT 379 379-397 gaAfgAfuCfaUfuUfuCfuCf 1249 GfGfcUfgAfgAfaAfaUfgAfuC 1250 aGfcCfdTsdT fuUfcdTsdT 380 380-398 aaGfaUfcAfuUfuUfcUfcAf 1251 AfGfgCfuGfaGfaAfaAfuGfaU 1252 gCfcUfdTsdT fcUfudTsdT 385 385-403 caUfuUfuCfuCfaGfcCfuCf 1253 UfCfuGfgAfgGfcUfgAfgAfaA 1254 cAfgAfdTsdT faUfgdTsdT 394 394-412 agCfcUfcCfaGfaGfgAfuGf 1255 UfGfaAfcAfuCfcUfcUfgGfaG 1256 uUfcAfdTsdT fgCfudTsdT 396 396-414 ccUfcCfaGfaGfgAfuGfuUf 1257 AfUfuGfaAfcAfuCfcUfcUfgG 1258 cAfaUfdTsdT faGfgdTsdT 397 397-415 cuCfcAfgAfgGfaUfgUfuCf 1259 UfAfuUfgAfaCfaUfcCfuCfuG 1260 aAfuAfdTsdT fgAfgdTsdT 401 401-419 agAfgGfaUfgUfuCfaAfuAf 1261 CfAfgUfuAfuUfgAfaCfaUfcC 1262 aCfuGfdTsdT fuCfudTsdT 403 403-421 agGfaUfgUfuCfaAfuAfaCf 1263 CfAfcAfgUfuAfuUfgAfaCfaU 1264 uGfuGfdTsdT fcCfudTsdT 407 407-425 ugUfuCfaAfuAfaCfuGfuGf 1265 AfCfcUfcAfcAfgUfuAfuUfgA 1266 aGfgUfdTsdT faCfadTsdT 409 409-427 uuCfaAfuAfaCfuGfuGfaGf 1267 CfCfaCfcUfcAfcAfgUfuAfuU 1268 gUfgGfdTsdT fgAfadTsdT 410 410-428 ucAfaUfaAfcUfgUfgAfgGf 1269 AfCfcAfcCfuCfaCfaGfuUfaUf 1270 uGfgUfdTsdT uGfadTsdT 411 411-429 caAfuAfaCfuGfuGfaGfgUf 1271 GfAfcCfaCfcUfcAfcAfgUfuA 1272 gGfuCfdTsdT fuUfgdTsdT 412 412-430 aaUfaAfcUfgUfgAfgGfuGf 1273 GfGfaCfcAfcCfuCfaCfaGfuUf 1274 gUfcCfdTsdT aUfudTsdT 413 413-431 auAfaCfuGfuGfaGfgUfgGf 1275 AfGfgAfcCfaCfcUfcAfcAfgU 1276 uCfcUfdTsdT fuAfudTsdT 414 414-432 uaAfcUfgUfgAfgGfuGfgUf 1277 AfAfgGfaCfcAfcCfuCfaCfaGf 1278 cCfuUfdTsdT uUfadTsdT 416 416-434 acUfgUfgAfgGfuGfgUfcCf 1279 CfCfaAfgGfaCfcAfcCfuCfaCf 1280 uUfgGfdTsdT aGfudTsdT 418 418-436 ugUfgAfgGfuGfgUfcCfuUf 1281 UfCfcCfaAfgGfaCfcAfcCfuCf 1282 gGfgAfdTsdT aCfadTsdT 419 419-437 guGfaGfgUfgGfuCfcUfuGf 1283 UfUfcCfcAfaGfgAfcCfaCfcUf 1284 gGfaAfdTsdT cAfcdTsdT 425 425-443 ugGfuCfcUfuGfgGfaAfuUf 1285 UfCfcAfaAfuUfcCfcAfaGfgA 1286 uGfgAfdTsdT fcCfadTsdT 431 431-449 uuGfgGfaAfuUfuGfgAfaAf 1287 GfUfaAfuUfuCfcAfaAfuUfcC 1288 uUfaCfdTsdT fcAfadTsdT 432 432-450 ugGfgAfaUfuUfgGfaAfaUf 1289 GfGfuAfaUfuUfcCfaAfaUfuC 1290 uAfcCfdTsdT fcCfadTsdT 433 433-451 ggGfaAfuUfuGfgAfaAfuUf 1291 AfGfgUfaAfuUfuCfcAfaAfuU 1292 aCfcUfdTsdT fcCfcdTsdT 434 434-452 ggAfaUfuUfgGfaAfaUfuAf 1293 UfAfgGfuAfaUfuUfcCfaAfaU 1294 cCfuAfdTsdT fuCfcdTsdT 458 458-476 agAfgGfaAfuUfaUfgAfuCf 1295 GfAfaAfgAfuCfaUfaAfuUfcC 1296 uUfuCfdTsdT fuCfudTsdT 459 459-477 gaGfgAfaUfuAfuGfaUfcUf 1297 GfGfaAfaGfaUfcAfuAfaUfuC 1298 uUfcCfdTsdT fcUfcdTsdT 463 463-481 aaUfuAfuGfaUfcUfuUfcCf 1299 AfGfaAfgGfaAfaGfaUfcAfuA 1300 uUfcUfdTsdT faUfudTsdT 464 464-482 auUfaUfgAfuCfuUfuCfcUf 1301 AfAfgAfaGfgAfaAfgAfuCfaU 1302 uCfuUfdTsdT faAfudTsdT 466 466-484 uaUfgAfuCfuUfuCfcUfuCf 1303 UfUfaAfgAfaGfgAfaAfgAfuC 1304 uUfaAfdTsdT faUfadTsdT 468 468-486 ugAfuCfuUfuCfcUfuCfuUf 1305 CfUfuUfaAfgAfaGfgAfaAfgA 1306 aAfaGfdTsdT fuCfadTsdT 471 471-489 ucUfuUfcCfuUfcUfuAfaAf 1307 GfGfuCfuUfuAfaGfaAfgGfaA 1308 gAfcCfdTsdT faGfadTsdT 476 476-494 ccUfuCfuUfaAfaGfaCfcAf 1309 UfGfgAfuGfgUfcUfuUfaAfg 1310 uCfcAfdTsdT AfaGfgdTsdT 477 477-495 cuUfcUfuAfaAfgAfcCfaUf 1311 CfUfgGfaUfgGfuCfuUfuAfaG 1312 cCfaGfdTsdT faAfgdTsdT 479 479-497 ucUfuAfaAfgAfcCfaUfcCf 1313 UfCfcUfgGfaUfgGfuCfuUfuA 1314 aGfgAfdTsdT faGfadTsdT 481 481-499 uuAfaAfgAfcCfaUfcCfaGf 1315 CfCfuCfcUfgGfaUfgGfuCfuU 1316 gAfgGfdTsdT fuAfadTsdT 482 482-500 uaAfaGfaCfcAfuCfcAfgGf 1317 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fcUfgdTsdT 979 979-997 gaGfaGfcGfaCfuGfcCfuGf 1553 AfGfaCfcAfgGfcAfgUfcGfcU 1554 gUfcUfdTsdT fcUfcdTsdT 980 980-998 agAfgCfgAfcUfgCfcUfgGf 1555 CfAfgAfcCfaGfgCfaGfuCfgC 1556 uCfuGfdTsdT fuCfudTsdT 981 981-999 gaGfcGfaCfuGfcCfuGfgUf 1557 GfCfaGfaCfcAfgGfcAfgUfcG 1558 cUfgCfdTsdT fcUfcdTsdT 982  982-1000 agCfgAfcUfgCfcUfgGfuCf 1559 GfGfcAfgAfcCfaGfgCfaGfuC 1560 uGfcCfdTsdT fgCfudTsdT 983  983-1001 gcGfaCfuGfcCfuGfgUfcUf 1561 CfGfgCfaGfaCfcAfgGfcAfgU 1562 gCfcGfdTsdT fcGfcdTsdT 984  984-1002 cgAfcUfgCfcUfgGfuCfuGf 1563 GfCfgGfcAfgAfcCfaGfgCfaG 1564 cCfgCfdTsdT fuCfgdTsdT 989  989-1007 gcCfuGfgUfcUfgCfcGfcAf 1565 AfAfuUfuGfcGfgCfaGfaCfcA 1566 aAfuUfdTsdT fgGfcdTsdT 990  990-1008 ccUfgGfuCfuGfcCfgCfaAf 1567 GfAfaUfuUfgCfgGfcAfgAfcC 1568 aUfuCfdTsdT faGfgdTsdT 991  991-1009 cuGfgUfcUfgCfcGfcAfaAf 1569 GfGfaAfuUfuGfcGfgCfaGfaC 1570 uUfcCfdTsdT fcAfgdTsdT 992  992-1010 ugGfuCfuGfcCfgCfaAfaUf 1571 CfGfgAfaUfuUfgCfgGfcAfgA 1572 uCfcGfdTsdT fcCfadTsdT 994  994-1012 guCfuGfcCfgCfaAfaUfuCf 1573 CfUfcGfgAfaUfuUfgCfgGfcA 1574 cGfaGfdTsdT fgAfcdTsdT 995  995-1013 ucUfgCfcGfcAfaAfuUfcCf 1575 UfCfuCfgGfaAfuUfuGfcGfgC 1576 gAfgAfdTsdT faGfadTsdT 996  996-1014 cuGfcCfgCfaAfaUfuCfcGf 1577 GfUfcUfcGfgAfaUfuUfgCfgG 1578 aGfaCfdTsdT fcAfgdTsdT 997  997-1015 ugCfcGfcAfaAfuUfcCfgAf 1579 CfGfuCfuCfgGfaAfuUfuGfcG 1580 gAfcGfdTsdT fgCfadTsdT 999  999-1017 ccGfcAfaAfuUfcCfgAfgAf 1581 UfUfcGfuCfuCfgGfaAfuUfuG 1582 cGfaAfdTsdT fcGfgdTsdT 1004 1004-1022 aaUfuCfcGfaGfaCfgAfaGf 1583 GfUfgGfcUfuCfgUfcUfcGfgA 1584 cCfaCfdTsdT faUfudTsdT 1005 1005-1023 auUfcCfgAfgAfcGfaAfgCf 1585 CfGfuGfgCfuUfcGfuCfuCfgG 1586 cAfcGfdTsdT faAfudTsdT 1006 1006-1024 uuCfcGfaGfaCfgAfaGfcCf 1587 AfCfgUfgGfcUfuCfgUfcUfcG 1588 aCfgUfdTsdT fgAfadTsdT 1007 1007-1025 ucCfgAfgAfcGfaAfgCfcAf 1589 CfAfcGfuGfgCfuUfcGfuCfuC 1590 cGfuGfdTsdT fgGfadTsdT 1008 1008-1026 ccGfaGfaCfgAfaGfcCfaCf 1591 GfCfaCfgUfgGfcUfuCfgUfcU 1592 gUfgCfdTsdT fcGfgdTsdT 1010 1010-1028 gaGfaCfgAfaGfcCfaCfgUf 1593 UfUfgCfaCfgUfgGfcUfuCfgU 1594 gCfaAfdTsdT fcUfcdTsdT 1013 1013-1031 acGfaAfgCfcAfcGfuGfcAf 1595 UfCfcUfuGfcAfcGfuGfgCfuU 1596 aGfgAfdTsdT fcGfudTsdT 1014 1014-1032 cgAfaGfcCfaCfgUfgCfaAf 1597 GfUfcCfuUfgCfaCfgUfgGfcU 1598 gGfaCfdTsdT fuCfgdTsdT 1015 1015-1033 gaAfgCfcAfcGfuGfcAfaGf 1599 UfGfuCfcUfuGfcAfcGfuGfgC 1600 gAfcAfdTsdT fuUfcdTsdT 1016 1016-1034 aaGfcCfaCfgUfgCfaAfgGf 1601 GfUfgUfcCfuUfgCfaCfgUfgG 1602 aCfaCfdTsdT fcUfudTsdT 1040 1040-1058 ccCfcAfcUfcAfuGfcUfcUf 1603 UfUfgUfaGfaGfcAfuGfaGfuG 1604 aCfaAfdTsdT fgGfgdTsdT 1042 1042-1060 ccAfcUfcAfuGfcUfcUfaCf 1605 GfGfuUfgUfaGfaGfcAfuGfaG 1606 aAfcCfdTsdT fuGfgdTsdT 1044 1044-1062 acUfcAfuGfcUfcUfaCfaAf 1607 GfGfgGfuUfgUfaGfaGfcAfuG 1608 cCfcCfdTsdT faGfudTsdT 1047 1047-1065 caUfgCfuCfuAfcAfaCfcCf 1609 GfGfuGfgGfgUfuGfuAfgAfg 1610 cAfcCfdTsdT CfaUfgdTsdT 1071 1071-1089 ccAfgAfuGfgAfuGfuGfaAf 1611 GfGfgGfuUfcAfcAfuCfcAfuC 1612 cCfcCfdTsdT fuGfgdTsdT 1073 1073-1091 agAfuGfgAfuGfuGfaAfcCf 1613 UfCfgGfgGfuUfcAfcAfuCfcA 1614 cCfgAfdTsdT fuCfudTsdT 1074 1074-1092 gaUfgGfaUfgUfgAfaCfcCf 1615 CfUfcGfgGfgUfuCfaCfaUfcC 1616 cGfaGfdTsdT faUfcdTsdT 1075 1075-1093 auGfgAfuGfuGfaAfcCfcCf 1617 CfCfuCfgGfgGfuUfcAfcAfuC 1618 gAfgGfdTsdT fcAfudTsdT 1077 1077-1095 ggAfuGfuGfaAfcCfcCfgAf 1619 GfCfcCfuCfgGfgGfuUfcAfcA 1620 gGfgCfdTsdT fuCfcdTsdT 1078 1078-1096 gaUfgUfgAfaCfcCfcGfaGf 1621 UfGfcCfcUfcGfgGfgUfuCfaC 1622 gGfcAfdTsdT faUfcdTsdT 1080 1080-1098 ugUfgAfaCfcCfcGfaGfgGf 1623 UfUfuGfcCfcUfcGfgGfgUfuC 1624 cAfaAfdTsdT faCfadTsdT 1084 1084-1102 aaCfcCfcGfaGfgGfcAfaAf 1625 UfGfuAfuUfuGfcCfcUfcGfgG 1626 uAfcAfdTsdT fgUfudTsdT 1085 1085-1103 acCfcCfgAfgGfgCfaAfaUf 1627 CfUfgUfaUfuUfgCfcCfuCfgG 1628 aCfaGfdTsdT fgGfudTsdT 1087 1087-1105 ccCfgAfgGfgCfaAfaUfaCf 1629 AfGfcUfgUfaUfuUfgCfcCfuC 1630 aGfcUfdTsdT fgGfgdTsdT 1088 1088-1106 ccGfaGfgGfcAfaAfuAfcAf 1631 AfAfgCfuGfuAfuUfuGfcCfcU 1632 gCfuUfdTsdT fcGfgdTsdT 1089 1089-1107 cgAfgGfgCfaAfaUfaCfaGf 1633 AfAfaGfcUfgUfaUfuUfgCfcC 1634 cUfuUfdTsdT fuCfgdTsdT 1096 1096-1114 aaAfuAfcAfgCfuUfuGfgUf 1635 UfGfgCfaCfcAfaAfgCfuGfuA 1636 gCfcAfdTsdT fuUfudTsdT 1097 1097-1115 aaUfaCfaGfcUfuUfgGfuGf 1637 GfUfgGfcAfcCfaAfaGfcUfgU 1638 cCfaCfdTsdT faUfudTsdT 1098 1098-1116 auAfcAfgCfuUfuGfgUfgCf 1639 GfGfuGfgCfaCfcAfaAfgCfuG 1640 cAfcCfdTsdT fuAfudTsdT 1104 1104-1122 cuUfuGfgUfgCfcAfcCfuGf 1641 CfAfcGfcAfgGfuGfgCfaCfcA 1642 cGfuGfdTsdT faAfgdTsdT 1106 1106-1124 uuGfgUfgCfcAfcCfuGfcGf 1643 UfUfcAfcGfcAfgGfuGfgCfaC 1644 uGfaAfdTsdT fcAfadTsdT 1112 1112-1130 ccAfcCfuGfcGfuGfaAfgAf 1645 CfAfcUfuCfuUfcAfcGfcAfgG 1646 aGfuGfdTsdT fuGfgdTsdT 1116 1116-1134 cuGfcGfuGfaAfgAfaGfuGf 1647 GfGfgAfcAfcUfuCfuUfcAfcG 1648 uCfcCfdTsdT fcAfgdTsdT 1117 1117-1135 ugCfgUfgAfaGfaAfgUfgUf 1649 GfGfgGfaCfaCfuUfcUfuCfaC 1650 cCfcCfdTsdT fgCfadTsdT 1118 1118-1136 gcGfuGfaAfgAfaGfuGfuCf 1651 CfGfgGfgAfcAfcUfuCfuUfcA 1652 cCfcGfdTsdT fcGfcdTsdT 1119 1119-1137 cgUfgAfaGfaAfgUfgUfcCf 1653 AfCfgGfgGfaCfaCfuUfcUfuC 1654 cCfgUfdTsdT faCfgdTsdT 1120 1120-1138 guGfaAfgAfaGfuGfuCfcCf 1655 UfAfcGfgGfgAfcAfcUfuCfuU 1656 cGfuAfdTsdT fcAfcdTsdT 1121 1121-1139 ugAfaGfaAfgUfgUfcCfcCf 1657 UfUfaCfgGfgGfaCfaCfuUfcU 1658 gUfaAfdTsdT fuCfadTsdT 1122 1122-1140 gaAfgAfaGfuGfuCfcCfcGf 1659 AfUfuAfcGfgGfgAfcAfcUfuC 1660 uAfaUfdTsdT fuUfcdTsdT 1123 1123-1141 aaGfaAfgUfgUfcCfcCfgUf 1661 AfAfuUfaCfgGfgGfaCfaCfuU 1662 aAfuUfdTsdT fcUfudTsdT 1124 1124-1142 agAfaGfuGfuCfcCfcGfuAf 1663 UfAfaUfuAfcGfgGfgAfcAfcU 1664 aUfuAfdTsdT fuCfudTsdT 1125 1125-1143 gaAfgUfgUfcCfcCfgUfaAf 1665 AfUfaAfuUfaCfgGfgGfaCfaC 1666 uUfaUfdTsdT fuUfcdTsdT 1126 1126-1144 aaGfuGfuCfcCfcGfuAfaUf 1667 CfAfuAfaUfuAfcGfgGfgAfcA 1668 uAfuGfdTsdT fcUfudTsdT 1127 1127-1145 agUfgUfcCfcCfgUfaAfuUf 1669 AfCfaUfaAfuUfaCfgGfgGfaC 1670 aUfgUfdTsdT faCfudTsdT 1128 1128-1146 guGfuCfcCfcGfuAfaUfuAf 1671 CfAfcAfuAfaUfuAfcGfgGfgA 1672 uGfuGfdTsdT fcAfcdTsdT 1129 1129-1147 ugUfcCfcCfgUfaAfuUfaUf 1673 CfCfaCfaUfaAfuUfaCfgGfgG 1674 gUfgGfdTsdT faCfadTsdT 1130 1130-1148 guCfcCfcGfuAfaUfuAfuGf 1675 AfCfcAfcAfuAfaUfuAfcGfgG 1676 uGfgUfdTsdT fgAfcdTsdT 1132 1132-1150 ccCfcGfuAfaUfuAfuGfuGf 1677 UfCfaCfcAfcAfuAfaUfuAfcG 1678 gUfgAfdTsdT fgGfgdTsdT 1134 1134-1152 ccGfuAfaUfuAfuGfuGfgUf 1679 UfGfuCfaCfcAfcAfuAfaUfuA 1680 gAfcAfdTsdT fcGfgdTsdT 1136 1136-1154 guAfaUfuAfuGfuGfgUfgA 1681 UfCfuGfuCfaCfcAfcAfuAfaU 1682 fcAfgAfdTsdT fuAfcdTsdT 1137 1137-1155 uaAfuUfaUfgUfgGfuGfaCf 1683 AfUfcUfgUfcAfcCfaCfaUfaA 1684 aGfaUfdTsdT fuUfadTsdT 1138 1138-1156 aaUfuAfuGfuGfgUfgAfcAf 1685 GfAfuCfuGfuCfaCfcAfcAfuA 1686 gAfuCfdTsdT faUfudTsdT 1139 1139-1157 auUfaUfgUfgGfuGfaCfaGf 1687 UfGfaUfcUfgUfcAfcCfaCfaU 1688 aUfcAfdTsdT faAfudTsdT 1140 1140-1158 uuAfuGfuGfgUfgAfcAfgA 1689 GfUfgAfuCfuGfuCfaCfcAfcA 1690 fuCfaCfdTsdT fuAfadTsdT 1142 1142-1160 auGfuGfgUfgAfcAfgAfuCf 1691 CfCfgUfgAfuCfuGfuCfaCfcA 1692 aCfgGfdTsdT fcAfudTsdT 1145 1145-1163 ugGfuGfaCfaGfaUfcAfcGf 1693 GfAfgCfcGfuGfaUfcUfgUfcA 1694 gCfuCfdTsdT fcCfadTsdT 1147 1147-1165 guGfaCfaGfaUfcAfcGfgCf 1695 AfCfgAfgCfcGfuGfaUfcUfgU 1696 uCfgUfdTsdT fcAfcdTsdT 1148 1148-1166 ugAfcAfgAfuCfaCfgGfcUf 1697 CfAfcGfaGfcCfgUfgAfuCfuG 1698 cGfuGfdTsdT fuCfadTsdT 1149 1149-1167 gaCfaGfaUfcAfcGfgCfuCf 1699 GfCfaCfgAfgCfcGfuGfaUfcU 1700 gUfgCfdTsdT fgUfcdTsdT 1150 1150-1168 acAfgAfuCfaCfgGfcUfcGf 1701 CfGfcAfcGfaGfcCfgUfgAfuC 1702 uGfcGfdTsdT fuGfudTsdT 1151 1151-1169 caGfaUfcAfcGfgCfuCfgUf 1703 AfCfgCfaCfgAfgCfcGfuGfaU 1704 gCfgUfdTsdT fcUfgdTsdT 1152 1152-1170 agAfuCfaCfgGfcUfcGfuGf 1705 GfAfcGfcAfcGfaGfcCfgUfgA 1706 cGfuCfdTsdT fuCfudTsdT 1153 1153-1171 gaUfcAfcGfgCfuCfgUfgCf 1707 GfGfaCfgCfaCfgAfgCfcGfuG 1708 gUfcCfdTsdT faUfcdTsdT 1154 1154-1172 auCfaCfgGfcUfcGfuGfcGf 1709 CfGfgAfcGfcAfcGfaGfcCfgU 1710 uCfcGfdTsdT fgAfudTsdT 1155 1155-1173 ucAfcGfgCfuCfgUfgCfgUf 1711 UfCfgGfaCfgCfaCfgAfgCfcG 1712 cCfgAfdTsdT fuGfadTsdT 1156 1156-1174 caCfgGfcUfcGfuGfcGfuCf 1713 CfUfcGfgAfcGfcAfcGfaGfcC 1714 cGfaGfdTsdT fgUfgdTsdT 1157 1157-1175 acGfgCfuCfgUfgCfgUfcCf 1715 GfCfuCfgGfaCfgCfaCfgAfgC 1716 gAfgCfdTsdT fcGfudTsdT 1160 1160-1178 gcUfcGfuGfcGfuCfcGfaGf 1717 CfAfgGfcUfcGfgAfcGfcAfcG 1718 cCfuGfdTsdT faGfcdTsdT 1200 1200-1218 ggAfgGfaAfgAfcGfgCfgUf 1719 GfCfgGfaCfgCfcGfuCfuUfcC 1720 cCfgCfdTsdT fuCfcdTsdT 1201 1201-1219 gaGfgAfaGfaCfgGfcGfuCf 1721 UfGfcGfgAfcGfcCfgUfcUfuC 1722 cGfcAfdTsdT fcUfcdTsdT 1203 1203-1221 ggAfaGfaCfgGfcGfuCfcGf 1723 CfUfuGfcGfgAfcGfcCfgUfcU 1724 cAfaGfdTsdT fuCfcdTsdT 1204 1204-1222 gaAfgAfcGfgCfgUfcCfgCf 1725 AfCfuUfgCfgGfaCfgCfcGfuC 1726 aAfgUfdTsdT fuUfcdTsdT 1205 1205-1223 aaGfaCfgGfcGfuCfcGfcAf 1727 CfAfcUfuGfcGfgAfcGfcCfgU 1728 aGfuGfdTsdT fcUfudTsdT 1207 1207-1225 gaCfgGfcGfuCfcGfcAfaGf 1729 UfAfcAfcUfuGfcGfgAfcGfcC 1730 uGfuAfdTsdT fgUfcdTsdT 1208 1208-1226 acGfgCfgUfcCfgCfaAfgUf 1731 UfUfaCfaCfuUfgCfgGfaCfgC 1732 gUfaAfdTsdT fcGfudTsdT 1211 1211-1229 gcGfuCfcGfcAfaGfuGfuAf 1733 UfUfcUfuAfcAfcUfuGfcGfgA 1734 aGfaAfdTsdT fcGfcdTsdT 1212 1212-1230 cgUfcCfgCfaAfgUfgUfaAf 1735 CfUfuCfuUfaCfaCfuUfgCfgG 1736 gAfaGfdTsdT faCfgdTsdT 1213 1213-1231 guCfcGfcAfaGfuGfuAfaGf 1737 AfCfuUfcUfuAfcAfcUfuGfcG 1738 aAfgUfdTsdT fgAfcdTsdT 1214 1214-1232 ucCfgCfaAfgUfgUfaAfgAf 1739 CfAfcUfuCfuUfaCfaCfuUfgC 1740 aGfuGfdTsdT fgGfadTsdT 1215 1215-1233 ccGfcAfaGfuGfuAfaGfaAf 1741 GfCfaCfuUfcUfuAfcAfcUfuG 1742 gUfgCfdTsdT fcGfgdTsdT 1216 1216-1234 cgCfaAfgUfgUfaAfgAfaGf 1743 CfGfcAfcUfuCfuUfaCfaCfuU 1744 uGfcGfdTsdT fgCfgdTsdT 1217 1217-1235 gcAfaGfuGfuAfaGfaAfgUf 1745 UfCfgCfaCfuUfcUfuAfcAfcU 1746 gCfgAfdTsdT fuGfcdTsdT 1219 1219-1237 aaGfuGfuAfaGfaAfgUfgCf 1747 CfUfuCfgCfaCfuUfcUfuAfcA 1748 gAfaGfdTsdT fcUfudTsdT 1220 1220-1238 agUfgUfaAfgAfaGfuGfcGf 1749 CfCfuUfcGfcAfcUfuCfuUfaC 1750 aAfgGfdTsdT faCfudTsdT 1221 1221-1239 guGfuAfaGfaAfgUfgCfgAf 1751 CfCfcUfuCfgCfaCfuUfcUfuA 1752 aGfgGfdTsdT fcAfcdTsdT 1222 1222-1240 ugUfaAfgAfaGfuGfcGfaAf 1753 GfCfcCfuUfcGfcAfcUfuCfuU 1754 gGfgCfdTsdT faCfadTsdT 1223 1223-1241 guAfaGfaAfgUfgCfgAfaGf 1755 GfGfcCfcUfuCfgCfaCfuUfcU 1756 gGfcCfdTsdT fuAfcdTsdT 1224 1224-1242 uaAfgAfaGfuGfcGfaAfgGf 1757 AfGfgCfcCfuUfcGfcAfcUfuC 1758 gCfcUfdTsdT fuUfadTsdT 1225 1225-1243 aaGfaAfgUfgCfgAfaGfgGf 1759 AfAfgGfcCfcUfuCfgCfaCfuU 1760 cCfuUfdTsdT fcUfudTsdT 1226 1226-1244 agAfaGfuGfcGfaAfgGfgCf 1761 CfAfaGfgCfcCfuUfcGfcAfcU 1762 cUfuGfdTsdT fuCfudTsdT 1229 1229-1247 agUfgCfgAfaGfgGfcCfuUf 1763 CfGfgCfaAfgGfcCfcUfuCfgC 1764 gCfcGfdTsdT faCfudTsdT 1230 1230-1248 guGfcGfaAfgGfgCfcUfuGf 1765 GfCfgGfcAfaGfgCfcCfuUfcG 1766 cCfgCfdTsdT fcAfcdTsdT 1231 1231-1249 ugCfgAfaGfgGfcCfuUfgCf 1767 UfGfcGfgCfaAfgGfcCfcUfuC 1768 cGfcAfdTsdT fgCfadTsdT 1232 1232-1250 gcGfaAfgGfgCfcUfuGfcCf 1769 UfUfgCfgGfcAfaGfgCfcCfuU 1770 gCfaAfdTsdT fcGfcdTsdT 1233 1233-1251 cgAfaGfgGfcCfuUfgCfcGf 1771 UfUfuGfcGfgCfaAfgGfcCfcU 1772 cAfaAfdTsdT fuCfgdTsdT 1235 1235-1253 aaGfgGfcCfuUfgCfcGfcAf 1773 AfCfuUfuGfcGfgCfaAfgGfcC 1774 aAfgUfdTsdT fcUfudTsdT 1236 1236-1254 agGfgCfcUfuGfcCfgCfaAf 1775 CfAfcUfuUfgCfgGfcAfaGfgC 1776 aGfuGfdTsdT fcCfudTsdT 1237 1237-1255 ggGfcCfuUfgCfcGfcAfaAf 1777 AfCfaCfuUfuGfcGfgCfaAfgG 1778 gUfgUfdTsdT fcCfcdTsdT 1238 1238-1256 ggCfcUfuGfcCfgCfaAfaGf 1779 CfAfcAfcUfuUfgCfgGfcAfaG 1780 uGfuGfdTsdT fgCfcdTsdT 1239 1239-1257 gcCfuUfgCfcGfcAfaAfgUf 1781 AfCfaCfaCfuUfuGfcGfgCfaA 1782 gUfgUfdTsdT fgGfcdTsdT 1241 1241-1259 cuUfgCfcGfcAfaAfgUfgUf 1783 UfUfaCfaCfaCfuUfuGfcGfgC 1784 gUfaAfdTsdT faAfgdTsdT 1261 1261-1279 ggAfaUfaGfgUfaUfuGfgUf 1785 AfUfuCfaCfcAfaUfaCfcUfaUf 1786 gAfaUfdTsdT uCfcdTsdT 1262 1262-1280 gaAfuAfgGfuAfuUfgGfuG 1787 AfAfuUfcAfcCfaAfuAfcCfuA 1788 faAfuUfdTsdT fuUfcdTsdT 1263 1263-1281 aaUfaGfgUfaUfuGfgUfgAf 1789 AfAfaUfuCfaCfcAfaUfaCfcUf 1790 aUfuUfdTsdT aUfudTsdT 1264 1264-1282 auAfgGfuAfuUfgGfuGfaAf 1791 UfAfaAfuUfcAfcCfaAfuAfcC 1792 uUfuAfdTsdT fuAfudTsdT 1266 1266-1284 agGfuAfuUfgGfuGfaAfuUf 1793 UfUfuAfaAfuUfcAfcCfaAfuA 1794 uAfaAfdTsdT fcCfudTsdT 1267 1267-1285 ggUfaUfuGfgUfgAfaUfuUf 1795 CfUfuUfaAfaUfuCfaCfcAfaU 1796 aAfaGfdTsdT faCfcdTsdT 1289 1289-1307 caCfuCfuCfcAfuAfaAfuGf 1797 GfUfaGfcAfuUfuAfuGfgAfg 1798 cUfaCfdTsdT AfgUfgdTsdT 1313 1313-1331 uuAfaAfcAfcUfuCfaAfaAf 1799 CfAfgUfuUfuUfgAfaGfuGfu 1800 aCfuGfdTsdT UfuAfadTsdT 1320 1320-1338 cuUfcAfaAfaAfcUfgCfaCf 1801 GfGfaGfgUfgCfaGfuUfuUfuG 1802 cUfcCfdTsdT faAfgdTsdT 1321 1321-1339 uuCfaAfaAfaCfuGfcAfcCf 1803 UfGfgAfgGfuGfcAfgUfuUfu 1804 uCfcAfdTsdT UfgAfadTsdT 1322 1322-1340 ucAfaAfaAfcUfgCfaCfcUf 1805 AfUfgGfaGfgUfgCfaGfuUfuU 1806 cCfaUfdTsdT fuGfadTsdT 1323 1323-1341 caAfaAfaCfuGfcAfcCfuCf 1807 GfAfuGfgAfgGfuGfcAfgUfu 1808 cAfuCfdTsdT UfuUfgdTsdT 1324 1324-1342 aaAfaAfcUfgCfaCfcUfcCfa 1809 UfGfaUfgGfaGfgUfgCfaGfuU 1810 UfcAfdTsdT fuUfudTsdT 1328 1328-1346 acUfgCfaCfcUfcCfaUfcAf 1811 CfCfaCfuGfaUfgGfaGfgUfgC 1812 gUfgGfdTsdT faGfudTsdT 1332 1332-1350 caCfcUfcCfaUfcAfgUfgGf 1813 AfUfcGfcCfaCfuGfaUfgGfaG 1814 cGfaUfdTsdT fgUfgdTsdT 1333 1333-1351 acCfuCfcAfuCfaGfuGfgCf 1815 GfAfuCfgCfcAfcUfgAfuGfgA 1816 gAfuCfdTsdT fgGfudTsdT 1335 1335-1353 cuCfcAfuCfaGfuGfgCfgAf 1817 GfAfgAfuCfgCfcAfcUfgAfuG 1818 uCfuCfdTsdT fgAfgdTsdT 1338 1338-1356 caUfcAfgUfgGfcGfaUfcUf 1819 GfUfgGfaGfaUfcGfcCfaCfuG 1820 cCfaCfdTsdT faUfgdTsdT 1344 1344-1362 ugGfcGfaUfcUfcCfaCfaUf 1821 CfAfgGfaUfgUfgGfaGfaUfcG 1822 cCfuGfdTsdT fcCfadTsdT 1345 1345-1363 ggCfgAfuCfuCfcAfcAfuCf 1823 GfCfaGfgAfuGfuGfgAfgAfuC 1824 cUfgCfdTsdT fgCfcdTsdT 1346 1346-1364 gcGfaUfcUfcCfaCfaUfcCf 1825 GfGfcAfgGfaUfgUfgGfaGfaU 1826 uGfcCfdTsdT fcGfcdTsdT 1347 1347-1365 cgAfuCfuCfcAfcAfuCfcUf 1827 CfGfgCfaGfgAfuGfuGfgAfgA 1828 gCfcGfdTsdT fuCfgdTsdT 1348 1348-1366 gaUfcUfcCfaCfaUfcCfuGf 1829 CfCfgGfcAfgGfaUfgUfgGfaG 1830 cCfgGfdTsdT faUfcdTsdT 1353 1353-1371 ccAfcAfuCfcUfgCfcGfgUf 1831 UfGfcCfaCfcGfgCfaGfgAfuG 1832 gGfcAfdTsdT fuGfgdTsdT 1354 1354-1372 caCfaUfcCfuGfcCfgGfuGf 1833 AfUfgCfcAfcCfgGfcAfgGfaU 1834 gCfaUfdTsdT fgUfgdTsdT 1355 1355-1373 acAfuCfcUfgCfcGfgUfgGf 1835 AfAfuGfcCfaCfcGfgCfaGfgA 1836 cAfuUfdTsdT fuGfudTsdT 1357 1357-1375 auCfcUfgCfcGfgUfgGfcAf 1837 UfAfaAfuGfcCfaCfcGfgCfaG 1838 uUfuAfdTsdT fgAfudTsdT 1360 1360-1378 cuGfcCfgGfuGfgCfaUfuUf 1839 CfCfcUfaAfaUfgCfcAfcCfgGf 1840 aGfgGfdTsdT cAfgdTsdT 1361 1361-1379 ugCfcGfgUfgGfcAfuUfuAf 1841 CfCfcCfuAfaAfuGfcCfaCfcGf 1842 gGfgGfdTsdT gCfadTsdT 1362 1362-1380 gcCfgGfuGfgCfaUfuUfaGf 1843 AfCfcCfcUfaAfaUfgCfcAfcCf 1844 gGfgUfdTsdT gGfcdTsdT 1363 1363-1381 ccGfgUfgGfcAfuUfuAfgGf 1845 CfAfcCfcCfuAfaAfuGfcCfaCf 1846 gGfuGfdTsdT cGfgdTsdT 1366 1366-1384 guGfgCfaUfuUfaGfgGfgUf 1847 AfGfuCfaCfcCfcUfaAfaUfgCf 1848 gAfcUfdTsdT cAfcdTsdT 1369 1369-1387 gcAfuUfuAfgGfgGfuGfaCf 1849 AfGfgAfgUfcAfcCfcCfuAfaA 1850 uCfcUfdTsdT fuGfcdTsdT 1370 1370-1388 caUfuUfaGfgGfgUfgAfcUf 1851 AfAfgGfaGfuCfaCfcCfcUfaA 1852 cCfuUfdTsdT faUfgdTsdT 1371 1371-1389 auUfuAfgGfgGfuGfaCfuCf 1853 GfAfaGfgAfgUfcAfcCfcCfuA 1854 cUfuCfdTsdT faAfudTsdT 1372 1372-1390 uuUfaGfgGfgUfgAfcUfcCf 1855 UfGfaAfgGfaGfuCfaCfcCfcU 1856 uUfcAfdTsdT faAfadTsdT 1373 1373-1391 uuAfgGfgGfuGfaCfuCfcUf 1857 GfUfgAfaGfgAfgUfcAfcCfcC 1858 uCfaCfdTsdT fuAfadTsdT 1374 1374-1392 uaGfgGfgUfgAfcUfcCfuUf 1859 UfGfuGfaAfgGfaGfuCfaCfcC 1860 cAfcAfdTsdT fcUfadTsdT 1404 1404-1422 ucUfgGfaUfcCfaCfaGfgAf 1861 CfAfgUfuCfcUfgUfgGfaUfcC 1862 aCfuGfdTsdT faGfadTsdT 1408 1408-1426 gaUfcCfaCfaGfgAfaCfuGf 1863 UfAfuCfcAfgUfuCfcUfgUfgG 1864 gAfuAfdTsdT faUfcdTsdT 1409 1409-1427 auCfcAfcAfgGfaAfcUfgGf 1865 AfUfaUfcCfaGfuUfcCfuGfuG 1866 aUfaUfdTsdT fgAfudTsdT 1411 1411-1429 ccAfcAfgGfaAfcUfgGfaUf 1867 GfAfaUfaUfcCfaGfuUfcCfuG 1868 aUfuCfdTsdT fuGfgdTsdT 1412 1412-1430 caCfaGfgAfaCfuGfgAfuAf 1869 AfGfaAfuAfuCfcAfgUfuCfcU 1870 uUfcUfdTsdT fgUfgdTsdT 1419 1419-1437 acUfgGfaUfaUfuCfuGfaAf 1871 GfGfuUfuUfcAfgAfaUfaUfcC 1872 aAfcCfdTsdT faGfudTsdT 1426 1426-1444 auUfcUfgAfaAfaCfcGfuAf 1873 CfCfuUfuAfcGfgUfuUfuCfaG 1874 aAfgGfdTsdT faAfudTsdT 1427 1427-1445 uuCfuGfaAfaAfcCfgUfaAf 1875 UfCfcUfuUfaCfgGfuUfuUfcA 1876 aGfgAfdTsdT fgAfadTsdT 1430 1430-1448 ugAfaAfaCfcGfuAfaAfgGf 1877 AfUfuUfcCfuUfuAfcGfgUfuU 1878 aAfaUfdTsdT fuCfadTsdT 1431 1431-1449 gaAfaAfcCfgUfaAfaGfgAf 1879 GfAfuUfuCfcUfuUfaCfgGfuU 1880 aAfuCfdTsdT fuUfcdTsdT siRNA Sequence with Chemical Modification Info lower case (n) = 2′-O—Me; Nf = 2′-F; dT = deoxy-T residue; s = phosphorothioate backbone modification; iB = inverted abasic

Example 2. Evaluation of In Vitro Potency of Anti-EGFR siRNAs

Each of the anti-EGFR siRNAs in Table 4 were transfected in each of three human non-small cell lung cancer (NSCLC) cell lines with the indicated EGFR mutational status:

-   -   H358: wild-type     -   H1650: Exon19 ΔE746-A750 deletion     -   H1975: Exon21 L858R and Exon20 T790M

At a single final concentration of 5 nM, each siRNA was formulated with a commercially-available transfection reagent (Lipofectamine RNAiMAX, Life Technologies) according to the manufacturer's “forward transfection” instructions. Cells were plated 24 h prior to transfection in duplicate within 24-well tissue culture plates. At 24 h (H1650 and H1975) or 48 h (H358) post-transfection, RNA was harvested from cells in all wells using a Qiagen RNeasy® Plus Mini Kit or Stratec InviTrap® RNA Cell HTS96 kit. The concentration of each isolated RNA was determined via A260 measurement using a NanoDrop spectrophotometer. RNA samples were then reverse transcribed to cDNA using the High Capacity RNA to cDNA Kit (Life Technologies) according to the manufacturer's instructions. cDNA samples were then evaluated by qPCR using EGFR-specific probes with results normalized to endogenous (3-actin and quantified using the standard 2^(−ΔΔCt) method. EGFR mRNA levels normalized to expression in controls were determined.

TABLE 4 qPCR, qPCR, qPCR, H358, H1650, H1975, 5 nM 5 nM 5 nM SEQ SEQ % Rel % Rel % Rel Avidity sense strand ID antisense strand ID EGFR EGFR EGFR ID# sequence (5′-3′) NO: sequence (5′-3′) NO: mRNA mRNA mRNA R-1006 agUfuUfgCfcAfaG 1217 AfCfuCfgUfgCfcUfu 1218 7.80% 5.50% 12.20% fgCfaCfgAfgUfdTs GfgCfaAfaCfudTsdT dT R-1010 agCfaGfuCfuUfaUf 1373 AfUfaGfuUfaGfaUfa 1374 3.80% 3.50% 8.60% cUfaAfcUfaUfdTsdT AfgAfcUfgCfudTsdT R-1018 gcCfgUfgCfgGfuU 1447 UfGfuUfgCfuGfaAfc 1448 7.40% 6.50% 13.00% fcAfgCfaAfcAfdTs CfgCfaCfgGfcdTsdT dT R-1011 gaUfgCfaAfaUfaAf 1395 GfUfcCfgGfuUfuUfa 1396 9.70% 4.80% 10.10% aAfcCfgGfaCfdTsdT UfuUfgCfaUfcdTsdT R-1001 gaCfgAfcAfgGfcCf 1147 GfAfcGfaGfgUfgGfc 1148 95.40% 118.90% 109.30% aCfcUfcGfuCfdTsdT CfuGfuCfgUfcdTsdT R-1002 acGfaCfaGfgCfcAf 1149 CfGfaCfgAfgGfuGfg 1150 69.30% 98.10% 112.20% cCfuCfgUfcGfdTsdT CfcUfgUfcGfudTsdT R-1003 ccAfcCfuCfgUfcGf 1163 GfCfgGfaCfgCfcGfa 1164 59.10% 81.50% 92.40% gCfgUfcCfgCfdTsdT CfgAfgGfuGfgdTsdT R-1004 guCfgGfcGfuCfcG 1177 GfAfcUfcGfgGfcGfg 1178 93.40% 121.30% 123.40% fcCfcGfaGfuCfdTs AfcGfcCfgAfcdTsdT dT R-1005 ccAfcAfaCfcAfcCf 1193 CfCfgUfgCfgCfgGfu 1194 70.30% 90.80% 124.10% gCfgCfaCfgGfdTsdT GfgUfuGfuGfgdTsdT R-1007 uuUfgCfcAfaGfgC 1221 UfUfaCfuCfgUfgCfc 1222 73.40% 75.70% 107.10% faCfgAfgUfaAfdTs UfuGfgCfaAfadTsdT dT R-1008 auGfcCfuUfaGfcAf 1359 GfAfuAfaGfaCfuGfc 1360 8.50% 7.70% 30.60% gUfcUfuAfuCfdTs UfaAfgGfcAfudTsdT dT R-1009 uuAfgCfaGfuCfuU 1369 AfGfuUfaGfaUfaAfg 1370 10.50% 10.60% 25.70% faUfcUfaAfcUfdTs AfcUfgCfuAfadTsdT dT R-1012 auGfcAfaAfuAfaA 1397 AfGfuCfcGfgUfuUfu 1398 22.30% 20.40% 41.30% faCfcGfgAfcUfdTs AfuUfuGfcAfudTsdT dT R-1013 ugCfaAfaUfaAfaAf 1399 CfAfgUfcCfgGfuUfu 1400 22.80% 24.80% 50.00% cCfgGfaCfuGfdTsdT UfaUfuUfgCfadTsdT R-1014 agGfaAfaUfcCfuGf 1419 GfCfgCfcAfuGfcAfg 1420 17.50% 12.10% 24.30% cAfuGfgCfgCfdTs GfaUfuUfcCfudTsdT dT R-1015 ggAfaAfuCfcUfgC 1421 GfGfcGfcCfaUfgCfa 1422 41.30% 41.10% 66.00% faUfgGfcGfcCfdTs GfgAfuUfuCfcdTsdT dT R-1016 aaAfuCfcUfgCfaUf 1425 AfCfgGfcGfcCfaUfg 1426 89.90% 89.70% 94.40% gGfcGfcCfgUfdTs CfaGfgAfuUfudTsdT dT R-1017 ugCfaUfgGfcGfcCf 1433 AfAfcCfgCfaCfgGfc 1434 81.40% 99.20% 108.60% gUfgCfgGfuUfdTs GfcCfaUfgCfadTsdT dT R-1019 gcGfgUfuCfaGfcA 1453 AfGfgGfuUfgUfuGfc 1454 30.20% 24.70% 45.10% faCfaAfcCfcUfdTs UfgAfaCfcGfcdTsdT dT R-1020 agUfgGfcGfgGfaC 1475 CfUfgAfcUfaUfgUfc 1476 71.80% 60.50% 82.50% faUfaGfuCfaGfdTs CfcGfcCfaCfudTsdT dT R-1021 cuCfaGfcAfaCfaUf 1481 CfCfaUfcGfaCfaUfg 1482 61.70% 99.40% 91.10% gUfcGfaUfgGfdTs UfuGfcUfgAfgdTsdT dT R-1022 gaUfcCfaAfgCfuGf 1517 CfAfuUfgGfgAfcAfg 1518 26.40% 24.50% 58.60% uCfcCfaAfuGfdTsdT CfuUfgGfaUfcdTsdT R-1023 ccAfaGfcUfgUfcCf 1523 UfCfcCfaUfuGfgGfa 1524 61.30% 84.80% 99.00% cAfaUfgGfgAfdTs CfaGfcUfuGfgdTsdT dT R-1024 gcCfuGfgUfcUfgC 1565 AfAfuUfuGfcGfgCfa 1566 20.00% 15.30% 23.60% fcGfcAfaAfuUfdTs GfaCfcAfgGfcdTsdT dT R-1025 ccUfgGfuCfuGfcCf 1567 GfAfaUfuUfgCfgGfc 1568 81.40% 77.40% 95.00% gCfaAfaUfuCfdTsdT AfgAfcCfaGfgdTsdT R-1026 cuGfgUfcUfgCfcG 1569 GfGfaAfuUfuGfcGfg 1570 20.40% 24.20% 65.90% fcAfaAfuUfcCfdTs CfaGfaCfcAfgdTsdT dT R-1027 ugGfuCfuGfcCfgC 1571 CfGfgAfaUfuUfgCfg 1572 15.40% 11.30% 41.20% faAfaUfuCfcGfdTs GfcAfgAfcCfadTsdT dT R-1028 cuGfcCfgCfaAfaUf 1577 GfUfcUfcGfgAfaUfu 1578 39.00% 39.70% 15.60% uCfcGfaGfaCfdTsdT UfgCfgGfcAfgdTsdT R-1029 ugCfcGfcAfaAfuU 1579 CfGfuCfuCfgGfaAfu 1580 28.70% 18.90% 30.90% fcCfgAfgAfcGfdTs UfuGfcGfgCfadTsdT dT R-1030 ccCfcAfcUfcAfuGf 1603 UfUfgUfaGfaGfcAfu 1604 34.00% 34.30% 17.10% cUfcUfaCfaAfdTsdT GfaGfuGfgGfgdTsdT R-1031 agAfuGfgAfuGfuG 1613 UfCfgGfgGfuUfcAfc 1614 17.80% 11.20% 52.60% faAfcCfcCfgAfdTs AfuCfcAfuCfudTsdT dT R-1032 cuGfcGfuGfaAfgA 1647 GfGfgAfcAfcUfuCfu 1648 30.50% 13.40% 40.40% faGfuGfuCfcCfdTs UfcAfcGfcAfgdTsdT dT R-1033 gaAfgAfaGfuGfuC 1659 AfUfuAfcGfgGfgAfc 1660 11.10% 5.90% 46.80% fcCfcGfuAfaUfdTs AfcUfuCfuUfcdTsdT dT R-1034 auGfuGfgUfgAfcA 1691 CfCfgUfgAfuCfuGfu 1692 27.90% 34.70% 9.00% fgAfuCfaCfgGfdTs CfaCfcAfcAfudTsdT dT R-1035 ugGfuGfaCfaGfaU 1693 GfAfgCfcGfuGfaUfc 1694 59.00% 48.80% 82.20% fcAfcGfgCfuCfdTs UfgUfcAfcCfadTsdT dT R-1036 guGfaCfaGfaUfcAf 1695 AfCfgAfgCfcGfuGfa 1696 21.40% 27.10% 91.20% cGfgCfuCfgUfdTs UfcUfgUfcAfcdTsdT dT R-1037 ugAfcAfgAfuCfaC 1697 CfAfcGfaGfcCfgUfg 1698 99.70% 90.20% 94.40% fgGfcUfcGfuGfdTs AfuCfuGfuCfadTsdT dT R-1038 gaCfaGfaUfcAfcGf 1699 GfCfaCfgAfgCfcGfu 1700 25.60% 10.90% 88.30% gCfuCfgUfgCfdTs GfaUfcUfgUfcdTsdT dT R-1039 acAfgAfuCfaCfgGf 1701 CfGfcAfcGfaGfcCfg 1702 94.80% 98.50% 95.40% cUfcGfuGfcGfdTs UfgAfuCfuGfudTsdT dT R-1040 caGfaUfcAfcGfgCf 1703 AfCfgCfaCfgAfgCfc 1704 108.70% 98.10% 90.00% uCfgUfgCfgUfdTs GfuGfaUfcUfgdTsdT dT R-1041 ucAfcGfgCfuCfgU 1711 UfCfgGfaCfgCfaCfg 1712 93.70% 76.20% 38.30% fgCfgUfcCfgAfdTs AfgCfcGfuGfadTsdT dT R-1042 caCfgGfcUfcGfuGf 1713 CfUfcGfgAfcGfcAfc 1714 86.90% 92.30% 92.70% cGfuCfcGfaGfdTsdT GfaGfcCfgUfgdTsdT R-1043 gaCfgGfcGfuCfcGf 1729 UfAfcAfcUfuGfcGfg 1730 60.00% 61.50% 8.00% cAfaGfuGfuAfdTs AfcGfcCfgUfcdTsdT dT R-1044 acGfgCfgUfcCfgCf 1731 UfUfaCfaCfuUfgCfg 1732 21.00% 36.60% 90.90% aAfgUfgUfaAfdTs GfaCfgCfcGfudTsdT dT R-1045 gcGfuCfcGfcAfaGf 1733 UfUfcUfuAfcAfcUfu 1734 32.20% 21.50% 101.60% uGfuAfaGfaAfdTs GfcGfgAfcGfcdTsdT dT R-1046 aaGfuGfuAfaGfaA 1747 CfUfuCfgCfaCfuUfc 1748 15.80% 10.10% 34.90% fgUfgCfgAfaGfdTs UfuAfcAfcUfudTsdT dT R-1047 cgAfaGfgGfcCfuU 1771 UfUfuGfcGfgCfaAfg 1772 15.10% 10.00% 24.90% fgCfcGfcAfaAfdTs GfcCfcUfuCfgdTsdT dT R-1048 aaGfgGfcCfuUfgCf 1773 AfCfuUfuGfcGfgCfa 1774 105.00% 96.80% 86.90% cGfcAfaAfgUfdTs AfgGfcCfcUfudTsdT dT R-1049 acCfuCfcAfuCfaGf 1815 GfAfuCfgCfcAfcUfg 1816 89.50% 97.60% 102.90% uGfgCfgAfuCfdTs AfuGfgAfgGfudTsdT dT R-1050 cuCfcAfuCfaGfuGf 1817 GfAfgAfuCfgCfcAfc 1818 39.00% 24.80% 68.60% gCfgAfuCfuCfdTs UfgAfuGfgAfgdTsdT dT R-1051 gcCfgGfuGfgCfaU 1843 AfCfcCfcUfaAfaUfg 1844 31.90% 30.40% 81.30% fuUfaGfgGfgUfdTs CfcAfcCfgGfcdTsdT dT

A list of the relative normalized EGFR mRNA levels, expressed as a percentage of control cells, for the initial single-concentration testing in all three cell lines is presented in Table 4. Of the siRNA candidates tested, four (4) achieved ≥80% down-regulation in all three cell lines tested. Based upon results obtained from this initial single-concentration experiment, we selected a total of four (4) candidate siRNAs (hs ID #333, #603, #622, and #685) for multi-concentration testing, which allowed determination of IC₅₀ (i.e., the concentration required to reduce EGFR expression by 50%), in each of these three cell lines. Cells were transfected as above at concentrations starting from 5000 pM using serial dilutions. 48 hours post-transfection, RNA was harvested and normalized EGFR mRNA levels were quantified as described above. Results are presented in Table 5, and all four siRNAs had comparable potency in term of silencing the mRNA target.

TABLE 5 qPCR, qPCR, qPCR, Viability Viability, Avidity H358, H1650, H1975, H358, H1975, ID# IC50, pM IC50, pM IC50, pM IC50, pM IC50, pM R-1006 21 3.5 181 395 21 R-1010 17 7 131 R-1018 21 10.4 99 R-1011 30 10.6 103 195 24

hs ID #333 and #622 were evaluated to see if reduction in EGFR mRNA resulted in reduced cell viability. Knocking down EGFR mRNA is known to affect cell viability and proliferation, particularly in cell lines carrying EGFR activating mutations, such as H1975. 3000 H358 and H1975 cells were plated in 384-well plates and increasing concentrations of siRNA-#333 and #622 in the presence with RNAiMAX were added to the cell culture. At 4 days post transfection, new media and siRNAs with transfection reagent were added to the cell culture plates. After a total of seven days (3 days post 2^(nd) transfection), cell viability was assessed with CellTiter-Blue® (Promega), according to the manufacturer's instructions and is reported in table 2. Knocking down EGFR mRNA, as shown in table 2, had a dramatic effect on cell viability on both cell lines.

Next, an array of chemical modification patterns were introduced to siRNA-#333 (Table 6) and their effect on EGFR mRNA were tested in H358 and H1975 cells after transfection with RNAiMAX as described above. Modification patterns used in R-1068 and R-1105 caused significant activity loss while the others showed comparable IC₅₀ values to the original siRNA-#333 in both cell lines tested.

TABLE 6 qPCR, qPCR, SEQ SEQ H358, H1650, Avidity sense strand sequence ID antisense strand ID IC50, IC50, ID (5′-3′) NO: sequence (5′-3′) NO: pM pM R-1067 AGUUUGCCAAGGCA 1881 ACUCGUGCCUUG 1882 12.7 167.7 CGAGUdTsdT GCAAACUdTsdT R-1068 AaguuuGccAAGGcacGA 1883 ACUCGUGCCUUG 1884 304.8 >1000 GudTsdT GcAAACUdTsdT R-1102 iBagUfuUfgCfcAfaGfgC 1885 AfCfuCfgUfgCfcUfu 1886 18.7 95.3 faCfgAfgUfdTsdTiB GfgCfaAfaCfudTsdT R-1103 iBagUfuUfgCfcAfaGfgC 1887 AfsCfsusCfgUfgCfc 1888 48.4 99.1 faCfgAfgUfdTsdTiB UfuGfgCfaAfaCfudT sdT R-1104 iBagUfuUfgCfcAfaGfgC 1889 aCfuCfgUfgCfcUfuG 1890 60.5 145.9 faCfgAfgUfdTsdTiB fgCfaAfaCfudTsdT R-1105 iBagUfuUfgCfcAfaGfgC 1891 asCfsusCfgUfgCfcUf 1892 167.4 546.7 faCfgAfgUfdTsdTiB uGfgCfaAfaCfudTsdT

While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby. 

What is claimed is:
 1. A polynucleic acid molecule that mediates RNA interference against EGFR, comprising a sense strand and an antisense strand that hybridizes to an EGFR target sequence selected from SEQ ID NOs: 1-376, wherein: the polynucleic acid molecule comprises the sense strand comprising i) two consecutive 2′-O-methyl modified nucleotides at the 5′-end and ii) eight consecutive repeats of A-B, wherein A is a 2′-fluoro modified nucleotide and B is a 2′-O-Me modified nucleotide, and iii) at least one phosphorothioate internucleotide linkage; the polynucleic acid molecule comprises from about 90% to about 100% modification; and the polynucleic acid molecule is from about 10 to about 50 nucleotides in length.
 2. The polynucleic acid molecule of claim 1, wherein the antisense strand comprises two consecutive 2′-fluoro modified nucleotides at the 5′-end.
 3. The polynucleic acid molecule of claim 1, wherein the sense strand comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA).
 4. The polynucleic acid molecule of claim 1, wherein the sense strand further comprises at least one inverted abasic moiety.
 5. The polynucleic acid molecule of claim 1, wherein the polynucleic acid molecule further comprises a phosphorodithioate linkage on the antisense strand.
 6. The polynucleic acid molecule of claim 1, wherein the polynucleic acid molecule is from about 10 to about 30 nucleotides in length.
 7. The polynucleic acid molecule of claim 1, wherein the polynucleic acid molecule is at least 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
 8. The polynucleic acid molecule of claim 1, wherein the polynucleic acid molecule comprises about 100% modification.
 9. The polynucleic acid molecule of claim 1, wherein the sense strand and the antisense strand are RNA molecules.
 10. The polynucleic acid molecule of claim 1, wherein the sense strand comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 377-1892.
 11. The polynucleic acid molecule of claim 1, wherein the antisense strand comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 377-1892.
 12. A pharmaceutical composition comprising: a) the polynucleic acid molecule of claim 1; and b) a pharmaceutically acceptable excipient.
 13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is formulated as a nanoparticle formulation.
 14. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is formulated for parenteral, oral, intranasal, buccal, rectal, or transdermal administration.
 15. A method of treating a disease or disorder in a patient in need thereof, comprising administering to the patient a composition comprising the polynucleic acid molecule of claim
 1. 16. The method of claim 15, wherein the disease or disorder is a cancer.
 17. The method of claim 16, wherein the cancer comprises a EGFR-associated cancer.
 18. The method of claim 16, wherein the cancer comprises bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, glioblastoma multiforme, head and neck cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, or thyroid cancer.
 19. The method of claim 16, wherein the cancer comprises acute myeloid leukemia, CLL, DLBCL, or multiple myeloma.
 20. A method of inhibiting the expression of EGFR gene in a primary cell of a patient, comprising administering the polynucleic acid molecule of claim 1 to the primary cell.
 21. The method of claim 20, wherein the method is an in vivo method.
 22. The method of claim 20, wherein the patient is a human.
 23. The polynucleic acid molecule of claim 1, wherein the polynucleic acid molecule comprises two inverted abasic moieties.
 24. The polynucleic acid molecule of claim 2, wherein the antisense strand comprises eight consecutive repeats of B-A.
 25. The polynucleic acid molecule of claim 24, wherein the eight repeats of B-A are adjacent to the two consecutive 2′-fluoro modified nucleotides.
 26. The polynucleic acid molecule of claim 1, wherein the sense strand comprises 5′-B-B-A-B-A-B-A-B-A-B-A-B-A-B-A-B-A-B-A-dTdT-3′.
 27. The polynucleic acid molecule of claim 1, wherein the polynucleic acid molecule has improved stability relative to an equivalent polynucleic acid molecule without the modification of at least three phosphorothioate internucleotide linkages.
 28. The polynucleic acid molecule of claim 1, wherein the sense strand comprises a sequence selected from SEQ ID NOs: 377-1892.
 29. The polynucleic acid molecule of claim 1, wherein the antisense strand comprises a sequence selected from SEQ ID NOs: 377-1892.
 30. The polynucleic acid molecule of claim 1, wherein the polynucleic acid molecule comprises about 100% modification, and wherein at least 45% of the modification is 2′-O-methyl. 